Sperm-Specific Cation Channel, Catsper2, and Uses Therefor

ABSTRACT

Nucleic acid and protein sequences relating to a cation channel which is sperm-specific (CatSper2) are disclosed. The CatSper2 protein is shown to be specifically expressed in sperm. Nucleic acids, vectors, transformed cells, transgenic animals, polypeptides, and antibodies relating to the CatSper2 gene and protein are disclosed. Also provided are methods of in vitro fertilization and contraception, methods of identifying modulators of CatSper2 activity, methods of genotyping subjects with respect to CatSper2, methods of diagnosing and treating CatSper2-mediated disorders, including infertility, as well as methods of doing business related to CatSper2-mediated disorders.

RELATED APPLICATIONS

This application is a continuation application of and claims benefit ofpriority to U.S. patent application Ser. No. 14/249,990, filed on Apr.10, 2014, which is a divisional application of and claims benefit ofpriority to U.S. patent application Ser. No. 12/021,474, filed Jan. 29,2008 (now U.S. Pat. No. 8,729,248), which is a divisional application ofand claims benefit of priority to U.S. application Ser. No. 10/828,975,filed Apr. 21, 2004 (now U.S. Pat. No. 7,339,029), which is acontinuation of PCT/US02/33676, filed Oct. 22, 2002, which claimspriority to U.S. Provisional Application No. 60/345,324, filed Oct. 22,2001, all of which are hereby incorporated by reference in theirentirety.

GOVERNMENT SUPPORT

The present invention was made, in part, with support from NIH Grant HD36022. The U.S. government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the fields of molecular biology andreproductive technology. In particular, the invention relates to acation channel protein expressed specifically in sperm cells, nucleicacids encoding the protein, cells engineered to express the protein,assays for compounds affecting the activity of the protein, and the useof such compounds in the treatment or causation of infertility, or as ameans of contraception or animal control.

Description of the Related Art.

Sperm and ova reciprocally interact in mammalian fertilization(Wassarman et al. (2001), Nature Cell Biology 3:E59-E64; Yanagimachi(1994), in The Physiology of Reproduction, Knobil & Neill eds. (RavenPress, New York), pp. 189-315). To reach the site of fertilization,sperm must travel relatively long distances and become primed forfertilization of the ova through capacitation and other processes. Oncethey arrive at the surface of an ovum, sperm interact with ovumextracellular matrix glycoproteins including the zona pellucidaproteins. Sperm release acidic material during the acrosome reaction, asignaling event that presumably involves the opening of Ca²⁺ channelsand the influx of Ca²⁻ into the sperm heads (O'Toole et al. (2000), Mol.Biol. Cell 11:1571-84). The TRPC2 protein, a putative Ca²⁻-permeantchannel, has recently been implicated in the acrosome reaction(Jungnickel et al. (2001), Nat. Cell Biol. 3:499-502). Penetration ofsperm through the thick outer layer of the ovum is achieved throughchemical lysis of the ovum coat and/or the mechanical motion of sperm(Bedford (1998), Biol. Reprod. 59:1275-87). Following infiltration ofthe ovum zona pellucida coat, the sperm membrane fuses with that ofovum. Fusion is followed by activation of the fertilization process,beginning with Ca²⁺ oscillations in the ovum (Wassarman et al. (2001),supra; Yanagimachi (1994), in The Physiology of Reproduction, Knobil &Neill eds. (Raven Press, New York), pp. 189-315).

Ca²⁺ and cyclic nucleotides control sperm motility (Tash (1990), inControls of Sperm Motility: Biological and Clinical Aspects, Gagnon ed.(CRC Press, Boca Raton), pp. 229-240; Darszon et al. (1999), Physiol.Rev. 79:481-510; Hyne and Garbers (1979), Proc. Natl. Acad. Sci. USA76:5699-703) and several voltage-dependent Ca²⁺ channel (Ca_(v)) mRNAsand cyclic nucleotide-gated (CNG) channel proteins have been detected insperm cell precursors (Darszon et al. (1999), Physiol. Rev. 79:481-510;Serrano et al. (1999), FEBS Lett. 462:171-6; Weyand et al. (1994) Nature368:859-63; Wiesner et al. (1998), J. Cell Biol. 142:473-84).Furthermore, low voltage activated, dihydropyridine-sensitive “T-type”channels (Santi et al. (1996), Am. J. Physiol. 271:C1583-93; Arnoult etal. (1996), Proc. Natl. Acad. Sci. USA 93:13004-9) and pharmacologicallydefined N- and R-type currents have been measured in spermatogenic cells(Wennemuth et al. (2000). J. Biol. Chem. 275:21210-7). The role of thesechannels, however, in spermatogenesis or mature sperm function is notknown.

Recently, a voltage-gated channel (CatSperl) was discovered that isrequired for normal fertility of mice (Ren et al. (2001), Nature413:603-9).

SUMMARY OF THE INVENTION

In one aspect, the present invention provides isolated nucleic acidscomprising all or a portion of a CatSper2 gene. In some embodiments, theisolated nucleic acids include a nucleotide sequence of at least 10, 12,14, 16 or 18 consecutive nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3 orSEQ ID NO: 5, or a sequence complementary thereto. In other embodiments,the nucleic acids include nucleotide sequences encoding a CatSper2protein, at least a transmembrane domain of a CatSper2 protein, at leastan extracellular loop of a CatSper2 protein, at least a pore region of aCatSper2 protein, at least an epitope of a CatSper2 protein having highpredicted antigenicity, or a sequence complementary thereto. Inparticular embodiments, the nucleic acids include a sequence of SEQ IDNO: 1; a sequence of SEQ ID NO: 3; a sequence of SEQ ID NO: 5; asequence encoding a polypeptide comprising approximately residues104-126, 146-166, 176-195, 206-228, 241-262, and 316-340 of SEQ ID NO:2; a sequence encoding a polypeptide comprising approximately residues104-126, 146-166, 176-195, 206-228, 241-262, and 316-340 of SEQ ID NO:4; a sequence encoding a polypeptide comprising approximately residues102-124, 144-164, 174-193, 204-227, 239-260, and 314-338 of SEQ ID NO:6; a sequence encoding a polypeptide comprising approximately residues127-145, 196-205, and 263-315 of SEQ ID NO: 2; a sequence encoding apolypeptide comprising approximately residues 127-145, 196-205, and265-315 of SEQ ID NO: 4; a sequence encoding a polypeptide comprisingapproximately residues 125-143, 194-203, and 261-313 of SEQ ID NO: 6; asequence encoding a polypeptide comprising approximately residues280-303 of SEQ ID NO: 2; a sequence encoding a polypeptide comprisingapproximately residues 280-303 of SEQ ID NO: 4; a sequence encoding apolypeptide comprising approximately residues 278-301 of SEQ ID NO: 6; asequence encoding a polypeptide comprising approximately residues266-275, 386-400, 447-458, and 482-494 of SEQ ID NO: 2; a sequenceencoding a polypeptide comprising approximately residues 66-99, 266-275,and 394-414 of SEQ ID NO: 4; a sequence encoding a polypeptidecomprising approximately residues 64-89, 262-275 and 562-588 of SEQ IDNO: 6; and a sequence complementary thereto. In certain embodiments, theinvention provides nucleic acids comprising sequences that encode apolypeptide consisting essentially of approximately residues 266-275,386-400, 447-458, and 482-494 of SEQ ID NO: 2; approximately residues66-99, 266-275, and 394-414 of SEQ ID NO: 4; or approximately residues64-89, 262-275 and 562-588 of SEQ ID NO: 6; and sequences complementarythereto.

In another aspect, the invention provides isolated nucleic acidsencoding polypeptide having at least 80%, 85%, 90%, or 95% amino acidsequence identity with a CatSper2 protein; at least a transmembranedomain of a CatSper2 protein; at least an extracellular loop of aCatSper2 protein; and at least a pore region of a CatSper2 protein. Insome embodiments, the isolated nucleic acids encode a polypeptide havingat least 80%, 85%, 90% or 95% amino acid sequence identity with aCatSper2 protein and having CatSper2 activity in a cell capable ofexpressing CatSper2 activity.

In another aspect, the invention provides isolated nucleic acids thathybridize to at least a portion of a nucleic acid of SEQ ID NO: 1 or SEQID NO: 3 or SEQ ID NO: 5 under conditions including a wash step of1.0×SSC at 65° C., a wash step of 0.5×SSC, a wash step of 0.2×SSC, or awash step of 0.1×SSC. In some embodiments, the isolated nucleic acidsencode a polypeptide having CatSper2 activity.

In another aspect, the invention provides nucleic acid comprising anucleotide sequence encoding a polypeptide having CatSper2 activity, andthat hybridizes to at least a portion of a nucleic acid of SEQ ID NO: 1or SEQ ID NO: 3 or SEQ ID NO: 5 under conditions including a wash stepof 1.0×SSC at 65° C., a wash step of 0.5×SSC, a wash step of 0.2×SSC, ora wash step of 0.1×SSC; and that is operably joined to a heterologousregulatory region such that the sequence is expressed. In anotherembodiment, the invention provides a nucleic acid comprising anucleotide sequence encoding a polypeptide having at least 80%, 85%, 90%or 95% amino acid sequence identity with an amino acid sequence of SEQID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6; and is operably joined to aheterologous regulatory region such that the sequence is expressed.

In another aspect, the invention provides a kit for detecting at least aportion of a CatSper2 nucleic acid. The kits can include any of theforegoing isolated nucleic acids of the invention, and a means fordetecting the isolated nucleic acid. In some embodiments, the means fordetecting the isolated nucleic acid includes a detectable label boundthereto and, in some embodiments, the means includes a labeled secondarynucleic acid which specifically hybridizes to the first isolated nucleicacid.

In another aspect, the invention provides a vector including any of theforegoing isolated nucleic acids of the invention. In some embodiments,the vector includes a genetic construct capable of expressing thenucleic acids of the invention. In some embodiments, the nucleic acidsof the invention are operably joined to an exogenous regulatory regionand, in some embodiments, the nucleic acids are operably joined toheterologous coding sequences to form a fusion vector. In someembodiments, the vector includes a CatSper2 regulatory region and, insome embodiments, the CatSper2 regulatory region is operably joined to aheterologous coding sequence.

In another aspect, the invention provides cells transformed with theforegoing nucleic acids of the invention, or a genetic construct capableof expressing a nucleic acid of the invention. In some embodiments, thenucleic acid of the invention is operably joined to heterologous codingsequences to encode a fusion protein. In some embodiments, the cells arebacterial cells, yeast cells, insect cells, nematode cells, amphibiancells, rodent cells, or human cells. In some embodiments, the cells aremammalian somatic cells, fetal cells, embryonic stem cells, zygotes,gametes, germ line cells and transgenic animal cells.

In another aspect, the invention provides non-human transgenic animals.In these aspects, a genetic construct has introduced a modification intoa genome of the animal, or an ancestor of the animal, and themodification includes insertion of a nucleic acid encoding at least afragment of a CatSper2 protein, inactivation of an endogenous CatSper2gene, or insertion by homologous recombination of a reporter geneoperably joined to CatSper2 regulatory elements. In some embodiments,the modification is insertion of nucleic acid encoding a CatSper2protein, at least a transmembrane domain of a CatSper2 protein, at leastan extracellular loop of a CatSper2 protein, at least a pore region of aCatSper2 protein, or at least an epitope of a CatSper2 protein havinghigh predicted antigenicity. In some embodiments, the animals are rats,mice, hamsters, guinea pigs, rabbit, dogs, cats, goats, sheep, pigs, andnon-human primates.

In another aspect, the invention provides substantially pure proteinpreparations including polypeptides selected from a CatSper2 protein; atleast a transmembrane domain of a CatSper2 protein; at least anextracellular loop of a CatSper2 protein; at least a pore region of aCatSper2 protein; and at least an epitope of a CatSper2 protein havinghigh predicted antigenicity. In particular embodiments, the polypeptideis selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; a sequenceencoding a polypeptide comprising approximately residues 104-126,146-166, 176-195, 206-228, 241-262, and 316-340 of SEQ ID NO: 2; asequence encoding a polypeptide comprising approximately residues104-126, 146-166, 176-195, 206-228, 241-262, and 316-340 of SEQ ID NO:4; a sequence encoding a polypeptide comprising approximately residues102-124, 144-164, 174-193, 204-227, 239-260, and 314-338 of SEQ ID NO:6; approximately residues 127-145, 196-205, and 263-315 of SEQ ID NO: 2;approximately residues 127-145, 196-205, and 265-315 of SEQ ID NO: 4;approximately residues 125-143, 194-203, and 261-313 of SEQ ID NO: 6;approximately residues 280-303 of SEQ ID NO: 2; approximately residues280-303 of SEQ ID NO: 4; approximately residues 278-301 of SEQ ID NO: 6;approximately residues 266-275, 386-400, 447-458, and 482-494 of SEQ IDNO: 2; approximately residues 66-99, 266-275, and 394-414 of SEQ ID NO:4; and approximately residues 64-89, 262-275 and 562-588 of SEQ ID NO:6. In certain embodiments, the invention provides substantially pureprotein preparations including polypeptides consisting essentially ofapproximately residues 266-275, 386-400, 447-458, and 482-494 of SEQ IDNO: 2; approximately residues 66-99, 266-275, and 394-414 of SEQ ID NO:4; or approximately residues 64-89, 262-275 and 562-588 of SEQ ID NO: 6.

In another aspect, the invention provides a substantially pure proteinpreparation including polypeptides having at least 80%, 85%, 90%, or 95%amino acid sequence identity with a CatSper2 protein; at least atransmembrane domain of a CatSper2 protein; at least an extracellularloop of a CatSper2 protein; or at least a pore region of a CatSper2protein. In some embodiments, the substantially pure protein preparationincludes a polypeptide having at least 80%, 85%, 90%, or 95% amino acidsequence identity with a CatSper2 protein and having CatSper2 activityin a cell capable of expressing CatSper2 activity.

In another aspect, the invention provides a substantially pure antibodypreparation including an antibody raised against a CatSper2 epitope. Insome embodiments, the epitope has high predicted antigenicity. In someembodiments, the epitope includes an amino acid sequence selected fromwithin approximately residues 266-275, 386-400, 447-458, and 482-494 ofSEQ ID NO: 2; approximately residues 66-99, 266-275, and 394-414 of SEQID NO: 4; or approximately residues 64-89, 262-275 and 562-588 of SEQ IDNO: 6. In some embodiments, the antibody is a monoclonal antibody. Insome embodiments, the antibody is an Fab fragment, an F(ab')2 fragment,an Fv fragment, or a single-chain Fv fragment (scFv).

In another aspect, the invention provides a kit for detecting at leastan epitope of a CatSper2 protein. The kits include an anti-CatSper2antibody of the invention and a means for detecting said antibody. Insome embodiments, the means for detecting said anti-CatSper2 antibodyincludes a detectable label bound thereto and, in some embodiments, themeans for detecting said anti-CatSper2 antibody includes a labeledsecondary antibody which specifically binds to the anti-CatSper2antibody.

In another aspect, the invention provides methods of identifyingpotential modulators of CatSper2 activity. The methods includecontacting a candidate compound with a cell expressing a CatSper2protein; measuring an indicator of CatSper2 activity in the cell;determining whether the candidate compound caused an increase ordecrease in the indicator relative to a reference level; and identifyingthe candidate compound as a potential modulator of CatSper2 activity ifthe compound causes an increase or decrease in the indicator. In someembodiments, the indicator is an indicator of the level of mRNA encodingthe CatSper2 protein, an indicator of the level of CatSper2 protein, anindicator of cation flux across a membrane of said cell, or an indicatorof whole cell or channel currents of said cell. In some embodiments, thecell has been transformed with a genetic construct capable of expressinga CatSper2 protein. In some embodiments, the cell is a mature sperm celland the indicator is a measure of sperm motility.

In another aspect, the invention provides methods of identifying apotential modulator of CatSper2 activity comprising contacting acandidate compound with at least a structural domain of a CatSper2protein; measuring binding, if any, between the candidate compound andthe CatSper2 moiety; and identifying the candidate compound as apotential modulator of CatSper2 activity if the binding is significant.In some embodiments, the CatSper2 moiety is a CatSper2 protein; at leasta transmembrane domain of a CatSper2 protein, at least an extracellularloop of a CatSper2 protein; or at least a pore region of a CatSper2protein.

In another aspect, the invention provides a method of decreasing thefertility of a male subject by administering a compound to the subjectwhich decreases CatSper2 activity. In another aspect, the inventionprovides a method of causing reversible infertility in a male subject byadministering a compound to the subject which decreases CatSper2activity. In another aspect, the invention provides a method ofcontraception in which a compound which decreases CatSper2 activity isadministered to a male or female subject. In each of the foregoingembodiments, the compound can be in an injection, a transdermal patch, abioerodable implant, a lubricant, a moisturizer, a foam, a jelly, or asponge. If the subject is a female, the compound can be administeredinto at least one of the vagina, uterus or fallopian tubes. In each ofthe foregoing embodiments, the compound can be a nucleic acid which isantisense to at least a portion of a CatSper2 gene or an antibody to aCatSper2 protein, including an Fab fragment, an F(ab′)₂ fragment, an Fvfragment, or an scFv fragment. In some embodiments, the subject is amammal. In some embodiments, the subjects are humans, dogs, cats, cows,sheep, horses, mice, rats, raccoons, and gophers. In other embodiments,the subjects are fish, amphibians or insects. In related aspects, theinvention provides for the use of a compound which decreases CatSper2activity in the preparation of a medicament for decreasing the fertilityof a male subject, or causing reversible infertility in a male subject,or in the preparation of a contraceptive for administration to a male orfemale. Thus, the invention provides contraceptive preparationsincluding compounds which decrease CatSper2 activity, including nucleicacids which are antisense to at least a portion of a CatSper2 gene andantibodies to a CatSper2 protein.

In another aspect, the invention provides methods of diagnosing aCatSper2-related disorder in a mammal by determining the presence orabsence of a mutation in a CatSper2 gene. In some embodiments, thepresence or absence of differences between a determined nucleic acid oramino acid sequence and a reference sequence indicates the presence orabsence of mutations in the CatSper2 gene. In some embodiments, themethod includes contacting at least a fragment of the CatSper2 proteinwith an antibody known to bind to a CatSper2 protein in which a mutationis known to be present or absent and detecting binding between theantibody and the CatSper2 protein. In other embodiments, the methodincludes measuring an indicator of CatSper2 activity in a cell; andcomparing the measured indicator to a reference level. The indicator canbe an indicator of the level of mRNA encoding CatSper2 protein, anindicator of the level of CatSper2 protein, an indicator of cation fluxacross a membrane of said cell, or an indicator of whole cell or channelcurrents of said cell. In some embodiments, the disorder isCatSper2-related infertility. In another aspect, the invention providesmethods of genotyping a subject with respect to a CatSper2 gene.

In another aspect, the invention provides a method of in vitrofertilization by sperm having decreased CatSper2 activity, decreasedmotility, or decreased ability to penetrate a zona pellucida, in which azona pellucida is removed from at least one ovum; and the ovum iscontacted with at least one sperm.

In another aspect, a method of treating a subject characterized byinfertility due to decreased CatSper2 activity is provided. The methodincludes transforming sperm or sperm progenitors of the subject with agenetic construct capable of expressing a CatSper2 protein and usingtransformed sperm of said subject to fertilize an ovum. Alternatively,the method includes administering a CatSper2 protein to sperm or spermprogenitors of the subject.

In another aspect, the invention provides methods of diagnosing ananti-CatSper2 antibody-mediated infertility caused by anti-CatSper2antibodies present in a female urogenital tract. In another aspect,methods of treating an anti-CatSper2 antibody-mediated infertilitycaused by anti-CatSper2 antibodies present in a female urogenital tractare provided.

In another aspect, the invention provides methods of conducting a drugdiscovery business including (a) identifying, by an assay of theinvention, one or more agents which antagonize CatSper2 activity; (b)determining if an agent identified in step (a), or an analog thereof,inhibits at least one of sperm motility or egg penetrance; (c)conducting therapeutic profiling of an agent identified as an inhibitorin step (b) for efficacy and toxicity in one or more animal models; and(d) formulating a pharmaceutical preparation including one or moreagents identified in step (c) as having an acceptable therapeuticprofile. In some embodiments, the method further includes the step ofestablishing a system for distributing the pharmaceutical preparationfor sale, and optionally including establishing a sales group formarketing the pharmaceutical preparation.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawing is illustrative of certain embodiments of theinvention but is not meant to limit the scope of the invention.

FIG. 1 (A) Amino acid sequence of murine CatSper2. The transmembranesegments and ion selective P region are underlined. The basic residuesof S4 are highlighted in bold. (B) Pair-wise alignment of CatSper2 andCatSperl transmembrane regions. The P regions are boxed.

DETAILED DESCRIPTION

The present invention depends, in part, upon the identification,isolation and characterization of a novel voltage-gated ion channel(CatSper2) expressed in male germ calls, but not in other tissuestested, and which plays a significant role in the motility of sperm andtheir ability to fertilize ova. The protein has been designated CatSper2to indicate that it is the second Cation channel which is Sperm-specificto be identified. The putative channel contains 6 transmembranesegments, a structure more similar to the voltage-gated potassiumchannels, but its ion selective pore indicates that it is a calciumchannel. The mRNA is expressed during the meiotic or post-meiotic stagesof spermatogenesis, and the protein is localized to the sperm flagellum,consistent with its role in the regulation of sperm motility. Therefore,inhibitors of the activity of the CatSper2 protein can be used as maleand female contraceptives.

The patent, scientific and medical publications referred to hereinestablish knowledge that was available to those of ordinary skill in theart at the time the invention was made. The entire disclosures of theissued U.S. patents, published and pending patent applications, andother references cited herein are hereby incorporated by reference. Inparticular, the entire disclosure of U.S. Provisional Patent Appin. Ser.No. 60/345,324, filed Oct. 22, 2001, is incorporated herein byreference.

Definitions.

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art; references to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent techniques which would be apparent to one of skill in theart. In order to more clearly and concisely describe the subject matterwhich is the invention, the following definitions are provided forcertain terms which are used in the specification.

As used herein, the term “CatSper2 protein” means a sperm-specificcation channel such as the human CatSper2 proteins disclosed in SEQ IDNO: 2 and SEQ ID NO: 4, human allelic and splice variants of thedisclosed CatSper2 proteins, non-human mammalian homologs of these humanCatSper2 proteins (e.g., SEQ ID NO: 6), and functional equivalentsthereof. The term CatSper2 protein refers to naturally occurringproteins as isolated from sperm, recombinantly produced proteins fromcells transformed with CatSper2 genes, and fusion proteins in whichCatSper2 sequences are fused to N-terminal or C-terminal polypeptides.The term “CatSper2 fragment” refers to fragments of the CatSper2proteins, such as structural domains and epitopes. A fragment of aCatSper2 protein comprises at least six amino acid residues.

As used herein, the term “CatSper2 gene” means a gene encoding aCatSper2 protein, including the human CatSper2 proteins disclosed in SEQID NO: 2 and SEQ ID NO: 4, human allelic and splice variants of thedisclosed CatSper2 proteins, non-human mammalian homologs of these humanCatSper2 proteins (e.g., SEQ ID NO: 6), and functional equivalentsthereof. The term CatSper2 gene refers to both naturally occurring genesas isolated from genomic DNA, and recombinantly produced genes in whichthe CatSper2 coding regions are operably joined to either endogenous orexogenous regulatory elements, with or without intron sequences, andwith or without 5′ or 3′-flanking sequences which can encodeheterologous (i.e., non-CatSper2) sequences to form a CatSper2 fusionprotein. A CatSper2 gene will include, at a minimum, a coding regionencoding the protein operably joined to regulatory elements (e.g.,promoter, enhancer) which allow transcription of the coding region tomRNA which can be translated into a CatSper2 protein.

As used herein “CatSper2” activity means any normal biological activityof a wild type CatSper2 protein when expressed in a cell or cell type inwhich CatSper2 is normally expressed and under conditions under whichCatSper2 is normally expressed. Such activity can include induction ofan ion current; mediation of cAMP-induced Ca²⁺ influx; restoration ofsperm motility when expressed in CatSper2-/-sperm; and/or restoration ofthe ability to penetrate eggs when expressed in CatSper2-/-sperm.CatSper2 activity can be measured in sperm cells or spermatocytes, or inother cells in which any necessary accessory factors are present.

As used herein with respect to nucleic acid and amino acid sequences,the term “identity” means a measure of the degree of similarity of twosequences based upon an alignment of the sequences which maximizesidentity and which is a function of the number of identical nucleotidesor residues, the number of total nucleotides or residues, and thepresence and length of gaps in the sequence alignment. A variety ofalgorithms and computer programs are available for determining sequenceidentity using standard parameters. For example, Gapped BLAST orPSI-BLAST (Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402),BLAST (Altschul et al. (1990), J. Mol Biol. 215:403-410), andSmith-Waterman (Smith et al. (1981), J. Mol. Biol. 147:195-197). Asstated herein, percent identity is based upon the default values for theBLAST algorithms.

As used herein, the term “homolog” means a protein which isevolutionarily-related to and shares substantial, conserved structuraland functional similarity with a reference protein, but which isnaturally present in a different species (e.g., human, rat and insectCatSper2 proteins are homologs of each other).

As used herein, the term “mutation” refers to a change in a nucleic acidsequence, whether or not expressed as a change in a correspondingencoded protein sequence, relative to some reference sequence. Thereference sequence can be a “wild-type” sequence (i.e., one or more highfrequency sequences in a population corresponding to a “normal”phenotype), or any other sequence. As used herein, the term mutation isintended to be synonymous with the term polymorphism, and therefore thedifferences between any two non-identical sequences can be regarded asmutations. The term mutation is intended to encompass insertions,deletions and/or substitutions of one or more nucleotides relative to areference sequence.

As used herein, the terms “exogenous” or “heterologous” mean, withrespect to two or more genetic sequences, that the genetic sequences donot occur in the same physical relation to each other in nature and/ordo not naturally occur within the same genome. For example, a geneticconstruct can include a coding region which is operably joined to one ormore regulatory elements, and these sequences are consideredheterologous to each other if they are not operably joined in natureand/or they are not found in the same genome in nature. Similarly, agenetic construct which is introduced into a cell is consideredheterologous to that cell to the extent that it contains geneticsequences not found in that cell. In addition, a synthetically-producedgenetic sequence based upon a naturally occurring sequence, will beheterologous to the naturally-occurring sequence to the extent thesequence has been altered and the synthetic sequence does not exist innature. Allelic variants of a sequence in a species are not consideredheterologous to each other.

As used herein, the term “operably joined” refers to a covalent andfunctional linkage of genetic regulatory elements and a genetic codingregion which can cause the coding region to be transcribed into mRNA byan RNA polymerase which can bind to one or more of the regulatoryelements. Thus, a regulatory region, including regulatory elements, isoperably joined to a coding region when RNA polymerase is capable underpermissive conditions of binding to a promoter within the regulatoryregion and causing transcription of the coding region into mRNA. In thiscontext, permissive conditions would include standard intracellularconditions for constitutive promoters, standard conditions and theabsence of a repressor or the presence of an inducer forrepressible/inducible promoters, and appropriate in vitro conditions, asknown in the art, for in vitro transcription systems.

As used herein, the term “expression” refers to the process by which acoding sequence of a gene is transcribed into a primary mRNA transcript,the primary mRNA transcript is processed into a mature mRNA, and themature mRNA is translated into a protein. Expression can optionallyinclude post-translation modifications of the resulting polypeptide.

As used herein, the phrase “genetic construct encoding a CatSper2protein” means a recombinant DNA, RNA, DNA-RNA hybrid, or nucleic acidanalog molecule which includes a genetic sequence encoding, or which iscomplementary to a genetic sequence encoding, or which is complementaryto a genetic sequence encoding, the amino acid sequence of the CatSper2protein, and which is capable of being expressed in a cell which hasbeen transformed with the construct. The construct can express theCatSper2 protein transiently, or can stably integrate into the genome ofthe cell and express the protein conditionally or constitutively.

As used herein, the term “vector” means any genetic construct, such asplasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.,which is capable of transferring gene sequences between cells. Vectorsare capable of one or more of replication, expression, and insertion orintegration, but need not possess each of these capabilities. Thus, theterm includes cloning, expression, homologous recombination, andknock-out vectors.

As used herein, with respect to genetic engineering, the term“transform” means to introduce into a cell or an organism an exogenousnucleic acid or nucleic acid analog which replicates within that cell ororganism, that encodes a polypeptide sequence which is expressed in thatcell or organism, and/or that is integrated into the genome of that cellor organism so as to affect the expression of a genetic locus. The term“transform” is used to embrace all of the various methods of introducingsuch nucleic acids or nucleic acid analogs, including, but not limitedto the methods referred to in the art as transformation, transfection,transduction, electroporation, ballistic injection, and the like.

As used herein, a “nucleic acid analog” means a molecule havingsufficient structural and functional similarity to a nucleic acid todirect sequence-specific forward or reverse transcription ofcomplementary nucleic acids, or to direct sequence-specific translationof an encoded polypeptide within a living cell. As used herein, wheneverthe term “nucleic acids” is used, the term is intended to embracenucleic acid analogs when such analogs would be useful or suitable inthe context of the usage.

As used herein, the term “reporter gene” means any genetic sequencewhich, when expressed, has a biochemical or phenotypic effect which isdetectable. Reporter genes are also known in the art as “marker” genes.

As used herein, the term “antibody” is intended to embrace naturallyproduced antibodies, recombinantly produced antibodies, and antibodyfragments such as Fab fragments, F(ab')2 fragments, Fv fragments, andsingle-chain Fv fragment (scFv).

As used herein, the term “effective amount” of an agonist or antagonist,or an enhancer or repressor, means the total amount of the activecomponent(s) of a composition that is sufficient to cause astatistically significant change of a detectable biochemical orphenotypic characteristic. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the effect, whetheradministered in combination, serially or simultaneously.

As used herein, the term “substantially pure” means a preparation whichcontains at least 60% (by dry weight) of the protein of interest,exclusive of the weight of other intentionally included compounds. Incertain embodiments, the preparation is at least 75%, at least 90%, orat least 99% the protein of interest by dry weight, exclusive of theweight of other intentionally included compounds. Purity can be measuredby any appropriate method, e.g., column chromatography, gelelectrophoresis, amino acid compositional analysis or HPLC analysis. Ifa preparation intentionally includes two or more different proteins ofthe invention, a “substantially pure” preparation means a preparation inwhich the total dry weight of the proteins of the invention is at least60% of the total dry weight, exclusive of the weight of otherintentionally included compounds. For preparations containing two ormore proteins of the invention, the total weight of the proteins of theinventions should be at least 75%, at least 90%, or at least 99%, of thetotal dry weight of the preparation, exclusive of the weight of otherintentionally included compounds. Thus, if the proteins of the inventionare mixed with one or more other compounds (e.g., diluents, stabilizers,detergents, excipients, salts, sugars, lipids) for purposes ofadministration, stability, storage, and the like, the weight of suchother compounds is ignored in the calculation of the purity of thepreparation.

As used herein, the terms “modulate” or “affect” mean to either increaseor decrease. As used herein, the terms “increase” and “decrease” mean,respectively, statistically significantly increase (i.e., p<0.1) andstatistically significantly decrease (i.e., p<0.1).

As used herein, the term “contacted” as in the phrase “A is contactedwith B,” means that A and B are brought into sufficient physicalproximity to interact at the molecular level, as by mixing A and Btogether in a solution, or pouring a solution of A over B on asubstrate. As used herein, the phrase “A is contacted with B” isintended to be equivalent to “B is contacted with A” and is not intendedto imply that either element is fixed relative to the other, or thateither element is moved relative to the other.

Numerical Ranges. As used herein, the recitation of a numerical rangefor a variable is intended to convey that the invention may be practicedwith the variable equal to any of the values within that range. Thus,for a variable which is inherently discrete, the variable can equal eachinteger value of the numerical range, including the end-points of therange. Similarly, for a variable which is inherently continuous, thevariable can equal each real value of the numerical range, including theend-points of the range. As an example, a variable which is described ashaving values between 0 and 2, can be 0, 1 or 2 for variables which areinherently discrete, and can be 0.0, 0.1, 0.01, 0.001, or any other realvalue ≦2 for variables which are inherently continuous.

Or. As used herein, unless specifically indicated otherwise, the word“or” is used in the “inclusive” sense of “and/or” and not the“exclusive” sense of “either/or.”

General Considerations.

The present invention depends, in part, upon the identification,isolation and characterization of a cation channel protein which isexpressed in sperm cells, but not in other tissues tested, and whichplays a significant role in the motility of sperm and their ability tofertilize ova. The protein has been designated CatSper2 to indicate thatit is the second Cation channel which is Sperm-specific to beidentified. The CatSper2 channel protein is present on the flagellumand, therefore, plays a role in sperm cell motility. Therefore,inhibitors of the activity of the CatSper2 protein can be used assperm-inhibiting contraceptives by men and/or women to cause temporary,reversible infertility.

The predicted CatSper2 ORF encodes a protein with six transmembranesegments (FIG. 1(A), SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5). Thesequences show similarities to the voltage-gated calcium channel family(Ca_(v)), and are most similar to CatSperl, another sperm cell-specificputative cation channel recently found to be important for motility andnormal fertilization (FIG. 1(B)) (Ren et al. (2001), supra). Thus,CatSper2 and CatSper1 define a new family of ion channels (Saier (2000),J. Membr. Biol. 175:165-80; Catterall (2000), Annu. Rev. Cell Dev. Biol.16:521-55). The features of this family include the presence of a singletransmembrane region with six membrane spanning segments and an apparentS4 voltage sensor, characteristics shared by the Kv and HCN channels,combined with a predicted calcium-selective pore region and an overallsequence similarity of the transmembrane region with the fourtransmembrane repeats in the voltage-gated calcium and sodium channels.

CatSper2 Nucleic Acids.

In one aspect, the present invention provides nucleic acid molecules, ornucleic acid analogs, encoding the CatSper2 proteins, or usefulfragments thereof. Two cDNAs of the human CatSper2 gene have beenidentified and are apparent splice variants of each other. Thesesequences are disclosed as SEQ ID NO:1 and SEQ ID NO: 3, and as GenbankAccession No. AF411817 and Genbank Accession No. AF411819, respectively.The full-length cDNA sequence of a murine homolog is disclosed as SEQ IDNO: 5 and as Genbank Accession No. AF411816.

Nucleic acid molecules of the invention can be DNA or RNA molecules, orhybrid DNA-RNA molecules. The nucleic acid analogs of the invention canbe any of those known in the art, such as peptide nucleic acids, analogsincluding modified bases (e.g., 2′-halogeno-2′-deoxynucleosides) and/oranalogs including modified internucleoside linkages (e.g.,phosphorothioate linkages), which are useful in applications such as invitro translation or antisense technologies. The nucleic acids can besense molecules corresponding to all or a portion of a CatSper2 genesequence, or can be antisense molecules which are complementary to allor a portion of a CatSper2 gene sequence. The nucleic acids can bederived from or correspond to genomic DNA or cDNA, or can be syntheticmolecules based upon a CatSper2 protein sequence and the genetic code(e.g., synthetic nucleic acids which reflect the codon usage preferencesin the host cells used in an expression system).

In some embodiments, the CatSper2 nucleic acids comprise the entirecoding region of a CatSper2 gene (e.g., SEQ ID NO: 1, SEQ ID NO: 3 orSEQ ID NO: 5). Such nucleic acids can be used to produce geneticconstructs for transformation of cells, or for in vitro transcriptionand translation systems. Such nucleic acids can also be used as probesin hybridization assays to detect CatSper2 sequences in samples of othernucleic acids.

In other embodiments, subsets of the CatSper2 nucleic acid sequences areprovided for use as primers for nucleic acid amplification reactions, asprobes in hybridization assays to detect CatSper2 sequences in samplesof other nucleic acids, or as probes to distinguish normal or wild-typesequences from abnormal or mutant sequences. In these embodiments, thenucleic acids of the invention comprise at least 10, 12, 14, 16 or 18consecutive nucleotides selected from a CatSper2 sequence such as SEQ IDNO: 1. Depending upon the nature of the application, it can bepreferable to choose CatSper2 sequences which will have unique targets,or which are expected to have unique targets, within a sample beingprobed or amplified. Thus, for example, sequences which are longer andsequences which do not include frequently repeated elements (forexample, polyadenylation signals) are more likely to he uniquelyrepresented within any given sample. For purposes of choosing primersfor amplification reactions, sequences of at least 15 nucleotides, andtypically 18-25 nucleotides, are used.

In certain embodiments, nucleic acids are provided which encodestructural domains of a CatSper2 protein, or which encode fragments ofthe protein which can serve as epitopes tor the generation ofantibodies. Thus for example, useful nucleic acids include thoseencoding the transmembrane domains of the CatSper2 proteins (i.e.,approximately residues 104-126, 146-166, 176-195, 206-228, 241-262, and316-340 of SEQ ID NO: 2, approximately residues 104-126, 146-166,176-195, 206-228, 241-262, and 316-340 of SEQ ID NO: 4, approximatelyresidues 102-124, 144-164. 174-193, 204-227, 239-260, and 314-338 of SEQID NO: 6, and allelic variants and homologs thereof), encoding theextracellular loops between transmembrane domains (i.e., 127-145,196-205, and 263-315 of SEQ ID NO: 2, 127-145, 196-205, and 265-315 ofSEQ ID NO: 4, 125-143, 194-203, and 261-313 of SEQ ID NO: 6, and allelicvariants and homologs thereof), or encoding the pore region (i.e., fromapproximately residue 280 to approximately residue 303 of SEQ ID NO: 2,from approximately residue 280 to approximately residue 303 of SEQ IDNO: 4, from approximately residue 278 to approximately residue 301 ofSEQ ID NO: 6, and allelic variants and homologs thereof). Other usefulnucleic acid acids include those encoding potential epitopes of theCatSper2 proteins, as identified by standard sequence analysistechniques described below. Thus, for example, useful nucleic acidsinclude those encoding the following human CatSper2 sequences: residues266-275, residues 386-400, residues 447-458, and residues 482-494 of SEQID NO: 2, residues 66-99, residues 266-275, and residues 394-414 of SEQID NO: 4, and residues 64-89, residues 262-275, and residues 562-588 ofSEQ ID NO: 6. Other useful epitopes include allelic and non-humanmammalian homologs of these epitopes.

In certain embodiments, nucleic acids are provided which encodepolypeptides have at least 80%, 85%, 90% or 95% amino acid sequenceidentity with at least a structural domain of a CatSper2 protein. Thus,in some embodiments, a nucleic acid is provided which encodes apolypeptide having at least 80%, 85%, 90% or 95% amino acid sequenceidentity with a transmembrane domain of a CatSper2 protein (e.g.,approximately residues 104-126, 146-166, 176-195, 206-228, 241-262, and316-340 of SEQ ID NO: 2; approximately residues 104-126, 146-166,176-195, 206-228, 241-262, and 316-340 of SEQ ID NO: 4; approximatelyresidues 102-124, 144-164, 174-193, 204-227, 239-260, and 314-338 of SEQID NO: 6; and allelic variants and homologs thereof), an extracellularloop between transmembrane domains (e.g., approximately residues127-145, 196-205, and 263-315 of SEQ ID NO: 2; approximately residues127-145, 196-205, and 265-315 of SEQ ID NO: 4; approximately residues125-143, 194-203, and 261-313 of SEQ ID NO: 6; and allelic variants andhomologs thereof), or a pore region (e.g., approximately residues280-303 of SEQ ID NO: 2; approximately residues 280-303 of SEQ ID NO: 4;approximately residues 278-301 of SEQ ID NO: 6; and allelic variants andhomologs thereof). In some embodiments, nucleic acids are providedencoding a polypeptide having at least 80%, 85%, 90% or 95% amino acidsequence identity with a CatSper2 protein and having CatSper2 activity.The ability of a protein to exhibit CatSper2 activity can be measured byits ability to complement a CatSper2-/-mutant (e.g., a CatSper2knock-out mutant) and restore a normal or CatSper2+/+ phenotype (e.g.,to restore sperm motility) in a cell otherwise capable of expressingCatSper2 activity (e.g., a sperm cell from the CatSper2-/-mutant).

In other embodiments, isolated nucleic acids are provided which includea nucleotide sequence that hybridizes to at least a portion of aCatSper2 coding sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ IDNO: 5) under stringent hybridization conditions. Such conditions includehybridizations employing a wash step of 1.0×SSC at 65° C., andequivalents thereof. More stringent conditions can include wash steps of0.5×SSC, 0.2×SSC, or even 0.1×SSC. Other equivalently stringentconditions are well known in the art. See, e.g., Ausubel et al., eds.(1989), Current Protocols in Molecular Biology, Vol. I, John Wiley &Sons, Inc., New York. In some embodiments, the nucleic acid encodes apolypeptide having CatSper2 activity.

In another aspect, the invention provides nucleic acids, either isolatedor existing within cells, in which a nucleotide sequence encoding apolypeptide having CatSper2 activity is operably joined to aheterologous regulatory region such that the CatSper2 sequence isexpressed. Thus, in certain embodiments, a heterologous regulatoryregion can be inserted into a chromosome such that it is operably joinedto an endogenous CatSper2 sequence. In some embodiments, the polypeptidehas at least 80%, 85%, 90% or 95% amino acid sequence identity with anamino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6. Inother embodiments, the nucleic acid encoding the polypeptide hybridizesto at least a portion of a nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 3or SEQ ID NO: 5 under conditions including a wash step of 1.0×SSC at 65°C., 0.5×SSC, 0.2×SSC, or 0.1×SSC.

In certain embodiments, the nucleic acids of the invention encodepolypeptides including a CatSper2 sequence of at least 50 amino acidresidues in length, or at least 100, 200 or 300 amino acid residues inlength. These polypeptides can include a CatSper2 sequence whichincludes at least one transmembrane domain, at least one extracellularloop domain, at least a pore region, or combinations thereof. In someembodiments, the polypeptide has CatSper2 activity. Such activity caninclude induction of ion current; mediation of cAMP-induced Ca²⁺ influx;restoration of sperm motility when expressed in CatSper2-/-sperm; and/orrestoration of the ability to fertilize eggs when expressed inCatSper2-/-sperm.

In another aspect, the invention provides kits for detecting at least aportion of a CatSper2 nucleic acid (i.e., CatSper2 genomic DNA, mRNA,cDNA or amplification products thereof). The kits include an isolatednucleic acid of the invention as a probe and means for demoting theprobe. The means for detecting the probe can be a detectable label boundto the probe or a secondary nucleic acid probe for detecting the firstprobe (e.g., labeled secondary nucleic acid which specificallyhybridizes to the isolated nucleic acid.).

Genetic Constructs.

In another aspect, the present invention provides genetic constructscomprising sequences selected from CatSper2 genes. In certainembodiments, the CatSper2 gene sequences are selected from the codingregion of the CatSper2 gene, and in other embodiments, the CatSper2 genesequences can be chosen from the CatSper2 regulatory regions extendingapproximately 1,000 bases 5′ of the transcription initiation codon, andextending approximately 1,000 bases 3′ of the termination codon.

In one series of embodiments, CatSper2 coding sequences (e.g., theentire coding region, sequences encoding structural domains, sequencesencoding potential epitopes, or sequences encoding useful primers orprobes) are operably joined to an endogenous or exogenous regulatoryregion to form an expression construct. Useful regulatory regions forthese purposes include the endogenous CatSper2 regulatory region,constitutive promoter sequences (e.g., CMV, SV40, EF2), and induciblepromoter sequences (e.g., lacZ, tet). Many useful vector systems arecommercially available. For example, useful bacterial vectors include,but are not limited to, pQE70, pQE60, pQE-9 (Qiagen, Valencia, Calif.),pBluescript II™ (Stratagene, La Jolla, Calif.), and pTRC99a, pKK223-3,pDR540 and pRIT2T (Pharmacia, Piscataway, N.J.), pTrc (Amann et al.(1988), Gene 69:301-315) and pET 11d (Studier et al. (1990), Methods inEnzymol. 185:60-89). Examples of vectors for expression in yeast includepYepSec1 (Baldari et al. (1987), EMBO J. 6:229-234), pMFa (Kurjan et al.(1982), Cell 30:933-943), pJRY88 (Schultz et al. (1987), Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). TheCatSper2 proteins can also be expressed to insect cells (e.g., Sf 9cells) using, for example, baculovirus expression vectors including, butnot limited to, pAc vectors (Smith et al. (1983), Mol. Cell Biol.3:2156-2165) and pVL vectors (Lucklow et al. (1989), Virology170:31-39). Examples of mammalian expression vectors include, but arenot limited to, pCDM8 (Seed (1987), Nature 329:840) and pMT2PC (Kaufmanet al. (1987), EMBO J. 6:187-195). Other useful eukaryotic vectorsinclude, but are not limited to, pXT1, pSG5 (Stratagene, La Jolla,Calif.), and pSVK3, pBPV, pMSG, and PSVLSV40 (Pharmacia, Piscataway,N.J.). Thus, one of ordinary skill in the art can choose a vector systemappropriate to the host cell to be transformed.

In other embodiments, the vectors comprise defective or partial CatSper2sequences in a “knock-out” vector. Such vectors are well-known in theart and can be used to produce a transgenic organism in which anendogenous gene is “knocked-out” by recombination with a partiallyhomologous exogenous sequence which introduces a mutation within theendogenous sequence. Typically, the vector is directed at an endogenoustarget sequence which can be all or part of a gene of interest. Thevector includes 5′ and 3′ flanking sequences which are homologous to the5′ and 3′ ends of the target. Between the 5′ and 3′ flanking sequencesis the sequence including the mutation. The mutation can be atermination mutation, frame-shift mutation, large deletion, or even theintroduction of a new coding sequence which serves both to disrupt theendogenous gene and to act as a marker to identify successful homologousrecombinants. Knock-out vectors are further discussed below.

In another series of embodiments, the CatSper2 coding sequences can bejoined to regulatory regions and exogenous coding sequences to form agenetic construct or fusion vector which encodes a fusion protein. Insome embodiments, the CatSper2 coding sequences can be joined toexogenous coding sequences that confer new and useful properties to thefusion protein. For example, fusion vectors and fusion proteins can beuseful to increase the expression of the CatSper2 protein, to increasethe solubility of the CatSper2 protein, or to aid in the purification ofthe CatSper2 protein (e.g., by providing a ligand sequence for affinitypurification). A proteolytic cleavage site can be introduced at thejunction of the CatSper2 and the non-CatSper2 protein sequences so thatthe CatSper2 protein can easily be separated from the fusion moiety.Typical fusion expression vectors include pGEX (Smith et al. (1988),Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to thetarget recombinant protein.

In another series of embodiments, genetic constructs are produced inwhich the coding region from a reporter gene is operably joined to theregulatory region of a CatSper2 gene. Such genetic constructs are usefulin assays to identify or characterize compounds that enhance or repressCatSper2 gene expression by enhancing or repressing transcription of theCatSper2 gene. A wide variety of suitable reporter genes are known tothose of skill in the art, and are commercially available. Examplesinclude, but are not limited to, the lacZ, luciferase and greenfluorescent protein genes.

Useful CatSper2 regulatory elements include sequences having at least80% nucleotide identity to at least 100-1,000, 200-800 or 300-700consecutive nucleotides selected from the 1,000 nucleotides immediately5′ to the CatSper2 transcription initiation site. Useful regulatoryelements retain the ability to promote transcription of a codingsequence operably joined to the element in a mammalian cell in which aCatSper2 gene is expressed. In particular, useful regulatory elementsretain the ability to promote transcription in cells in which theCatSper2 gene from which the element was derived is expressed, or inwhich a homolog of that CatSper2 gene is expressed.

Transformed Cell Lines.

In another aspect, the present invention provides cell lines transformedwith the nucleic acid molecules of the invention. Such cell lines cansimply propagate these nucleic acids (e.g., when transformed withcloning vectors) or can express the polypeptides encoded by thesenucleic acids (e.g., when transformed with expression vectors). Suchtransformed cell lines can be used to produce the CatSper2 proteins andCatSper2 fragments of the invention, or can be used in assays to screenfor compounds that enhance, repress, agonize, or antagonize CatSper2expression or activity.

The transformed cells can be produced by introducing into a cell anexogenous nucleic acid or nucleic acid analog which replicates withinthat cell, that encodes a polypeptide sequence which is expressed inthat cell, and/or that is integrated into the genome of that cell so asto affect the expression of a genetic focus. The transformation can beachieved by any of the standard methods referred to in the art astransformation, transfection, transduction, electroporation, ballisticinjection, and the like. The method of transformation is chosen to besuitable to the type of cells being transformed and the nature of thegenetic construct being introduced into the cells.

Useful cell lines for transformation include bacterial cells (e.g.,Escherichia coli), yeast cells (e.g., Saccharomyces cerevisiae), insectcells (e.g., Drosophila melanogaster Schneider cells), nematode cells(e.g., Caenorhabditis elegans), amphibian cells (e.g., Xenopus oocytes),rodent cells (e.g., Mus musculus (e.g., murine 3T3 fibroblasts), Rattusrattus, Chinese Hamster Ovary cells (e.g., CHO-K1)), and human cells(e.g., human skin fibroblasts, human embryonic kidney cells (e.g.,HEK-293 cells), COS cells). Although these and many other types of cellscan be transformed for purposes of producing the CatSper2 protein,preliminary studies have found that transformation of CHO-K1 and HEK-293cells does not result in detectable CatSper2 activity as determined bypatch-clamp measurements of channel currents. These latter cells appearto lack co-factors or accessory proteins present in sperm which arenecessary to CatSper2 activity, structural attributes of sperm which arenecessary for functional channel organization, or a necessary CatSper2post-translational processing or localization mechanism. Yeast twohybrid approaches and co-immunoprecipitation approaches can be used toscreen libraries to identify CatSper2 accessory, associating orinteracting proteins, including modulators of CatSper2 activity.

Appropriate cells can be transformed with any of the above-describedgenetic constructs in order to produce CatSper2 proteins, includingfragments of CatSper2 proteins, fusion proteins of CatSper2 proteins, ormarker proteins under the control of a CatSper2 regulatory region.

The cells can be transformed according to any method known in the artappropriate to the cell type being transformed. Appropriate methodsinclude those described generally in, e.g., Sambrook et al. (1989),Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratories,New York; and Davis el al. (1986), Basic Methods in Molecular Biology,Elsevier. Particular methods include calcium phosphate co-precipitation(Graham et al. (1973), Virol. 52:456-467), direct micro-injection intocultured cells (Capecchi (1980), Cell 22:479-488), electroporation(Shigekawa et al. (1988), BioTechniques 6:742-751), liposome-mediatedgene transfer (Mannino et al. (1988), BioTechniques 6:682-690),lipid-mediated transduction (Feigner et al. (1987), Proc. Natl. Acad.Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocitymicroprojectiles (Klein et al. (1987). Nature 327:70-73).

Transgenic Animals.

The present invention also provides for the production of transgenicnon-human animal models in which wild type, allelic variant, chimeric,or antisense CatSper2 sequences are expressed, or in which CatSper2sequences have been inactivated or deleted (e.g., “knock-out”constructs) or replaced with reporter or marker genes (e.g., “knock-inreporter” constructs). The CatSper2 sequences can be conspecific to thetransgenic animal (e.g., murine sequences in a transgenic mouse) ortranspecific to the transgenic animal (e.g. human sequence in atransgenic mouse). In such a transgenic animal, the transgenic sequencescan be expressed inducibly, constitutively or ectopically. Expressioncan be tissue-specific or organism-wide. Engineered expression ofCatSper2 sequences in tissues and cells not normally containing CatSper2gene products can cause novel alterations of cation flux and lead tonovel cell or tissue phenotypes. Ectopic or altered levels of expressionof CatSper2 sequences can alter cell, tissue and/or developmentalphenotypes. Transgenic animals are useful as models of disorders arisingfrom defects in CatSper2 activity.

Transgenic animals are also useful for screening compounds for theireffects on CatSper2 activity. Transgenic animals transformed withreporter constructs can be used to measure the transcriptional effectsof small molecules or drugs on physical perturbations on the expressionof CatSper2 genes and proteins in vivo. The transgenic animals of theinvention, can be used to screen such compounds for therapeutic utility.

Animal species suitable for use in the animal models of the presentinvention include, but are not limited to, rats, mice, hamsters, guineapigs, rabbits, dogs, cats, goats, sheep, pigs, and non-human primates(e.g., Rhesus monkeys, chimpanzees). For initial studies, transgenicrodents (e.g., mice) can be used due to their relative ease ofmaintenance and shorter life spans. Transgenic non-human primates can beused for longer term studies due to their greater similarity to humans.

Using the nucleic acids disclosed and otherwise enabled herein, thereare several embodiments of the creation of a transgenic animal. Thus,useful animal models include; (1) animals in which sequences encoding atleast a functional fragment of a CatSper2 gene has been recombinantlyintroduced into the genome of the animal as an additional gene, underthe regulation of either an exogenous or an endogenous promoter element,and as either a minigene (i.e., a genetic construct of the CatSper2 genebased on cDNA with introns removed) or a huge genomic fragment; (2)animals in which sequences encoding at least a functional fragment of aCatSper2 gene have beets recombinantly substituted for one or bothcopies of the animal's endogenous CatSper2 gene by homologousrecombination or gene targeting; (3) animals in which one or both copiesof one of the animal's homologous CatSper2 genes have been recombinantly“humanized” by the partial substitution of sequences encoding the humanhomolog by homologous recombination or gene targeting; (4) animals inwhich sequences encoding a reporter gene have replaced the endogenousCatSper2 gene by homologous recombination; (5) and “knock-out” animalsin which one or both copies of the animal's CatSper2 sequences have beenpartially or completely inactivated by the insertion, deletion orsubstitution of one or more nucleotides by homologous recombination.These and other transgenic animals of the invention are useful as modelsof infertility or other disorders arising from defects in the CatSper2gene and/or protein. These animals are also useful for screeningcompounds for their effects on the CatSper2 gene and/or protein.

To produce an animal model (e.g., a transgenic mouse), a wild type orallelic variant CatSper2 sequence or a wild type or allelic variant of arecombinant nucleic acid encoding at least a functional fragment of aCatSper2 protein can be inserted into a germ line or stem cell usingstandard techniques of oocyte or embryonic stem cell microinjection, orother methods of transformation of such cells. Alternatively, othercells from an adult organism can be employed. Animals produced by theseor similar processes are referred to as transgenic. Similarly, if it isdesired to inactivate or replace an endogenous CatSper2 sequence,homologous recombination using oocytes, embryonic stem or other cellscan be employed. Animals produced by these or similar processes arereferred to as “knock-out” (inactivation) or “knock-in” (replacement)models.

For oocyte injection, one or more copies of the recombinant DNAconstructs of the present invention can be inserted into the pronucleusof a just-fertilized oocyte. This oocyte is then reimplanted into apseudo-pregnant foster mother. The live born animals are screened forintegrants using standard DNA/mRNA analysis (e.g., from the tail veinsof offspring mice) for the presence of the inserted recombinanttransgene sequences. The transgene can be either a complete genomicsequence introduced into a host as part of a yeast artificial chromosome(YAC), bacterial artificial chromosome (BAC), or other chromosome DNAfragment; as a cDNA with either the endogenous promoter or aheterologous promoter; or as a minigene containing all of the codingregions and other elements found to be necessary for optimum expression.

To create a transgene, the target sequence of interest (e.g., a wildtype or allelic variant of a CatSper2 sequence) is typically ligatedinto a cloning site located downstream of a promoter element which willregulate the expression of RNA from the sequence. Downstream of thecoding sequence, there is typically a polyadenylation sequence. Analternative approach to creating a transgene is to use an exogenouspromoter and regulatory sequences to drive expression of the transgene.Finally, it is possible to create transgenes using large genomic DNAfragments such as YACs which contain the entire desired gene as well asits appropriate regulatory sequences.

Animal models can be created by targeting endogenous CatSper2 sequencesfor homologous recombination. These targeting events can have the effectof removing an endogenous sequence (knock-out) or altering theendogenous sequence to create an amino acid change associated with humandisease or an otherwise abnormal sequence (e.g., a sequence which ismore like the human sequence than the original animal sequence)(knock-in animal models). A large number of vectors are available toaccomplish such changes, and appropriate sources of genomic DNA formoose and other animals are commercially available (e.g., GenomeSystemsInc., St. Louis, Mo.).

The typical feature of these targeting vector constructs is that 2 to 4kb of genomic DNA is ligated 5′ to a selectable marker (e.g., abacterial neomycin resistance gene under its own promoter element termeda “neomycin cassette”). A second DNA fragment from the gene of interestis then ligated downstream of the neomycin cassette but upstream of asecond selectable marker (e.g., thymidine kinase). The DNA fragments arechosen such that mutant sequences can be introduced into the germ lineof the targeted animal by homologous replacement of the endogenoussequences by either one of the sequences included in the vector.Alternatively, the sequences can be chosen to cause deletion ofsequences that would normally reside between the left and right arms ofthe vector surrounding the neomycin cassette. The former is known as aknock-in, the latter is known as a knock-out.

Early embryos can also be infected to insert the recombinant DNAconstructs of the invention. In this method, the transgene (e.g., a wildtype or allelic variant of a CatSper2 sequence) is inserted into a viralor retroviral vector which is used to directly infect embryos (e.g.,mouse or non-human primate embryos) during the early stages ofdevelopment to generate partially transgenic animals. Some of thepartially transgenic animals will bear the transgenes in germ line cellsand can be bred to produce fully transgenic animals.

Alternatively, homologous recombination using a population of stem cellsallows for the screening of the population for successful transformants.Once identified, these can be injected into blastocysts, and aproportion of the resulting animals will show germ line transmission ofthe transgene. These partially transgenic animals can be bred to producefully transgenic animals.

Techniques of generating transgenic animals, as well as techniques forhomologous recombination or gene targeting, are now widely accepted andpracticed. A laboratory manual on the manipulation of the mouse embryo,for example, is available which details standard laboratory techniquesfor the production of transgenic mice (Hogan et al. (1986), Manipulatingthe Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). CatSper2 Proteins and Polypeptides.

In another aspect, the present invention provides substantially purepreparations of CatSper2 proteins. The proteins can be isolated fromsperm cells, using standard techniques such as immunoaffinitypurification with the antibodies of the invention (see below), or can beisolated from the transformed cells of the invention, in which they canbe expressed at higher levels and, optionally, as fusion proteins whichare more easily isolated and/or purified.

In some embodiments, the CatSper2 proteins comprise the entiretranslated sequence of the CatSper2 coding region. Examples of suchfull-length CatSper2 proteins include the human CatSper2 proteinsdisclosed as SEQ ID NO: 2 and SEQ ID NO: 4 and the mouse homologdisclosed as SEQ ID NO: 6, as well as allelic and non-human homologs ofCatSper2 proteins, and functional equivalents thereof.

In other embodiments, the CatSper2 proteins are CatSper2 fragments. Suchfragments include the structural domains of the CatSper2 proteins,including the transmembrane, loop and pore-forming regions of theproteins. Useful structural domains include the transmembrane domains ofthe human CatSper2 protein (i.e., approximately residues 104-126,146-166, 176-195, 206-228, 241-262, and 316-340 of SEQ ID NO: 2;approximately residues 104-126, 146-166, 176-195, 206-228, 241-262, and316-340 of SEQ ID NO: 4; approximately residues 102-124, 144-164,174-193, 204-227, 239-260, and 314-338 of SEQ ID NO: 6), theextracellular loops between transmembrane domains (i.e., approximatelyresidues 127-145, 196-205, and 263-315 of SEQ ID NO: 2; approximatelyresidues 127-145, 196-205, and 265-315 of SEQ ID NO: 4; approximatelyresidues 125-143, 194-203, and 261-313 of SEQ ID NO: 6), and the poreregion (i.e., approximately residues 280-303 of SEQ ID NO: 2;approximately residues 280-303 of SEQ ID NO: 4; approximately residues278-301 of SEQ ID NO: 6), as well as allelic variants and non-humanhomologs thereof. Other CatSper2 fragments include potentially usefulepitopes of the CatSper2 proteins, as identified by standard sequenceanalysis techniques. Thus, for example, useful CatSper2 fragmentsinclude the following human CatSper2 sequences: residues 266-275,386-400, 447-458 and 482-494 of SEQ ID NO: 2; residues 66-99, 266-275and 394-414 of SEQ ID NO: 4; and residues 64-89, 262-275 and 562-588 ofSEQ ID NO: 6.

In certain embodiments, polypeptides are provided having at least 80%,85%, 90% or 95% amino acid sequence identity with at least a structuraldomain of a CatSper2 protein. Thus, in some embodiments, a polypeptideis provided having at least 80%, 85%, 90% or 95% amino acid sequenceidentity with a transmembrane domain of a CatSper2 protein (e.g.,approximately residues 104-126, 146-166, 176-195, 206-228, 241-262, and316-340 of SEQ ID NO: 2; approximately residues 104-126, 146-166,176-195, 206-228, 241-262, and 316-340 of SEQ ID NO: 4; approximatelyresidues 102-124, 144-164, 174-193, 204-227, 239-260, and 314-338 of SEQID NO: 6, and allelic variants and homologs thereof), an extracellularloop between transmembrane domains (e.g., approximately residues127-145, 196-205, and 263-315 of SEQ ID NO: 2; approximately residues127-145, 196-205, and 265-315 of SEQ ID NO: 4; approximately residues125-143, 194-203, and 261-313 of SEQ ID NO: 6, and allelic variants andhomologs thereof), or a pore region (e.g., approximately residues280-303 of SEQ ID NO: 2, approximately residues 280-303 of SEQ ID NO: 4,approximately residues 278-301 of SEQ ID NO: 6, and allelic variants andhomologs thereof). In some embodiments, polypeptides are provided havingat least 80%, 85%, 90% or 95% amino acid sequence identity with aCatSper2 protein and having CatSper2 activity. The ability of a proteinto exhibit CatSper2 activity can be measured by its ability tocomplement a CatSper2-/-mutant (e.g., a CatSper2 knock-out mutant) andrestore a normal or CatSper2+/+ phenotype (e.g., to restore spermmotility) in a cell otherwise capable of expressing CatSper2 activity(e.g., a sperm cell from the CatSper2-/-mutant).

In certain embodiments, the polypeptides of the invention include aCatSper2 sequence of at least 50 amino acid residues in length, or atleast 100, 200 or 300 amino acid residues in length. These polypeptidescan include a CatSper2 sequence which includes at least onetransmembrane domain, at least one extracellular loop domain, at least apore region, or combinations thereof In some embodiments, thepolypeptide has CatSper2 activity. Such activity can include theinduction of ion currents when expressed in a cell (e.g., an oocyte);mediation of cAMP-induced Ca²⁺ influx; restoration of sperm motilitywhen expressed in CatSper2-/-sperm; and/or restoration of the ability topenetrate eggs when expressed in CatSper2-/-sperm.

Antibodies Against CatSper2 Proteins and Polypeptides.

In another aspect, the present invention provides substantially purepreparations of antibodies against the CatSper2 proteins, and methods ofmaking such antibodies. The antibodies can be polyclonal or monoclonal,and can be made by methods well known in the art. In particular, theinvention provides antibodies raised against CatSper2 epitopes havinghigh predicted antigenicity, which therefore will selectively bind toand, thereby, isolate or identify wild type and/or variant forms of theCatSper2 proteins.

The antibodies can be raised against the full-length CatSper2 proteins,against fragments of the CatSper2 proteins, or using any CatSper2epitopes which are characteristic of the proteins and whichsubstantially distinguish them from other proteins. In certainembodiments, the antibodies are raised against CatSper2 epitopesincluding, but not limited to, residues 266-275, 386-400, 447-458 and482-494 of SEQ ID NO: 2; residues 60-99, 266-275 and 394-414 of SEQ IDNO: 4; and residues 64-89, 262-275 and 562-588 of SEQ ID NO: 6. Otheruseful epitopes include allelic and non-human homologs of theseepitopes. Epitopes having a high predicted antigenicity were identifiedby prediction of hydrophobicity, surface probability and antigenic indexusing standard programs, including GCG and MacVector (Genetics ComputerGroup, University of Wisconsin Biotechnology Center, Madison, Wis.;Accelrys Inc., San Diego, Calif.), See also, Jameson and Wolf (1988),Comput. Appl. Biosci. 4:181-186.

CatSper2 immnnogen preparations can be produced from crude extracts(e.g., microsomal fractions of cells expressing the proteins), fromproteins or peptides substantially purified from cells which naturallyor recombinantly express them or, for small immunogens, by chemicalpeptide synthesis. The CatSper2 immunogens can also be in the form of afusion protein in which the non-CatSper2 portion is chosen for itsadjuvant properties and/or its ability to facilitate purification (e.g.,polyhistidine).

The antibodies of the invention can be polyclonal or monoclonal, or canbe antibody fragments, including Fab fragments, F(ab′)₂ fragments, Fvfragments, and single chain Fv fragments (scFv). In addition, afteridentifying useful antibodies by the method of the invention,recombinant antibodies can be generated, including any of the antibodyfragments listed above, as well as chimeric and/or humanized antibodiesbased upon non-human antibodies to the CatSper2 proteins. In light ofthe present disclosure of CatSper2 proteins, as well as thecharacterization of other CatSper2 proteins enabled herein, one ofordinary skill in the art can produce the above-described antibodies byany of a variety of standard means. For an overview of antibodytechniques, see Antibody Engineering, 2nd Ed., Borrebaek, ed., OxfordUniversity Press, Oxford (1995).

As a general matter, monoclonal anti-CatSper2 antibodies can be producedby first injecting a mouse, rabbit, goat or other suitable animal with aCatSper2 immunogen in a suitable carrier or diluent. Carrier proteins oradjuvants can be utilized, and booster injections (e.g., bi- ortri-weekly over 8-10 weeks) can be employed as necessary. After allowingfor development of a humoral response, the animals are sacrificed andtheir spleens are removed and resuspended in an appropriate buffer(e.g., phosphate buffered saline). The spleen cells serve as a source oflymphocytes, some of which will produce antibodies of the appropriatespecificity. These cells are then fused with an immortalized cell line(e.g., a myeloma), and the products of the fusion are plated into tissueculture wells in the presence of a selective agent (e.g., HAT). Thewells are serially screened and replated, selecting cells making auseful antibody each time. Typically, several screening and replatingprocedures are carried out until the wells contain single clones whichare positive for antibody production. Monoclonal antibodies produced bysuch clones can be purified by standard methods such as affinitychromatography using Protein A Sepharose, by ion-exchangechromatography, or by variations and combinations of these techniques.

The antibodies of the invention can be used in a variety ofapplications. For example, antibodies can be used in a purificationprocess (e.g., immunoaffinity purification) for CatSper2 proteins, inassays to detect the presence or level of CatSper2 proteins (e.g., in adiagnostic test for a CatSper2-related disorder), or in assays tomeasure the presence or level of CatSper2 expression in transformedcells (e.g., in assays for regulators of CatSper2 expression, in Westernblotting to identify cells expressing CatSper2 proteins, or inimmunocytochemistry or immunofluorescence techniques to establish thecellular or extracellular location of CatSper2 proteins).

The antibodies of the invention can be bound to or conjugated with othercompounds or materials for diagnostic and/or therapeutic uses. Forexample, they can be coupled to labels such as radionuclides,fluorescent compounds (e.g., rhodamine), or enzymes for imaging ortherapy. The labels can be bound to the antibodies covalently ornon-covalently.

In another aspect, the invention provides kits for detecting at least anepitope of a CatSper2 protein. The kits include an anti-CatSper2antibody and a means for detecting the antibody. The means for detectingthe antibody can be a detectable label bound to the antibody orsecondary antibodies for defecting the anit-CatSper2 antibodies (e.g., alabeled goat anti-rabbit-Ig antibody as a secondary antibody fordetecting a rabbit anti-CatSper2 antibody).

Assays for Modulators of CatSper2 Expression or Activity.

In another aspect, the present invention provides assays for modulatorsof CatSper2 expression or activity. The modulators can affect thetranscription, translation, post-translational processing, localization,or activity of a CatSper2 gene and/or protein.

Thus, in one series of embodiments, the transformed cells of theinvention are contacted with a candidate compound, and the effect of thecompound on the expression or activity of CatSper2 is determined. As ageneral matter, the assays require contacting a candidate compound witha cell expressing a CatSper2 protein and measuring an indicator ofCatSper2 activity in the cell. The indicator can be an indicator oftranscription (e.g., mRNA levels), translation (e.g., protein levels),post-translational processing (e.g., specific glycosylation),localization (e.g., immunohistochemistry), or activity (e.g., sodium orother monovalent ion flux; calcium or other divalent ion flux). Theindicator measurement is then compared to a reference level to determinewhether the candidate compound caused an increase or decrease in theindicator. The reference level can be extrinsic (e.g., a predeterminedbaseline level) or intrinsic (e.g., a measurement of the same cell priorto contact with the candidate compound). If an increase or decrease issignificant (based on a single reading or on multiple readings from oneor more cells), the candidate compound is identified as a potentialmodulator of CatSper2 activity. Assays for changes in CatSper2 activitycan include any of those used routinely in the art for other genes. Forexample, changes in the presence or levels of CatSper2 mRNA or proteincan be detected to identify enhancers or repressors of CatSper2expression. Alternatively, when using a reporter gene construct of theinvention, the biochemical or phenotypic change characteristic of thereporter can be used as an indication that the candidate compoundenhances or represses reporter gene expression. In other embodiments,changes in the activity of the CatSper2 protein can be detected bymeasuring, for example, the flux of cations mediated by the CatSper2protein, or by measuring whole cell or channel currents. Measurements ofion fluxes can be facilitated by the use of chromophores which changecolor depending upon the concentration of specific ions. The effects ofcandidate compounds on mature sperm cells can be tested to confirm orvalidate results obtained in the transformed cells of the invention.

Compounds which bind to CatSper2 are candidates for modulating CatSper2activity. Thus, in another series of embodiments, libraries of compoundscan be screened to identify candidates for modulating CatSper2 activityby contacting candidate compounds with a CatSper2 protein, or at least astructural domain of a CatSper2 protein, to identify compounds that bindto CatSper2. CatSper2 structural domains which can be used in thesemethods include transmembrane domains, and particularly extracellularloops and pore regions. In such methods, the CatSper2 protein orCatSper2 structural domain can be immobilized (e.g., on a column ormicroparticle) and a solution of the candidate compound can be contactedwith the CatSper2 moiety, or the candidate compound can be immobilized(e.g., on a column of microparticle) and a solution of the CatSperlmoiety can be contacted with the candidate compound. Alternatively, insome embodiments, neither the candidate compound nor the CatSper2 moietyis immobilized but, rather, both are in solution and binding is detectedby, for example, aggregation of particles bearing the binding partners.Binding can be detected by methods well known in the art (e.g.,radioactive or fluorescent labeling of one component of the potentialbinding pair; plasmon-resonance detection of binding; turbidity changesin aggregation assays). Compounds which, under physiological conditions(e.g., within the testis or epididymis, or within the vagina, uterus orfallopian tubes), exhibit significant binding (e.g., Ka <10 μM) to aCatSper2 protein, are potential modulators of CatSper2 activity.

Methods of Modulating Fertility.

The CatSper2 gene and proton are ideal targets for potentialcontraceptive drugs. Since the CatSper2 protein is expressed in theflagella of sperm, a specific blocker of CatSper2 can inhibit spermmotility, and thus can be effective as a contraceptive when used byeither sex. The restricted localization of CatSper2 to sperm andspermatogenic cells means that a specific blocker should not affectother tissues and thus side effects should be low or nonexistent.Finally, because the channel represents a novel structure, it can be anexcellent target for new channel agonists or antagonists.

Thus, in another aspect, the present invention provides methods ofdecreasing fertility by decreasing the expression or activity of aCatSper2 gene or protein. Such decreases in expression or activity canbe achieved by means of a small molecule that represses expression of aCatSper2 gene, by means of an antisense molecule that inhibits thetranslation of a CatSper2 mRNA, by means of a small molecule thatinterferes with CatSper2 translation or post-translational processing,by means of a small molecule that interferes with CatSper2 localization,by means of a molecule that blocks CatSper2 activity as an ion channel,or by any other molecule that decreases the expression of a CatSper2gene or hastens CatSper2 protein degradation. Antibodies, includingantibody fragments such as Fab, F(ab')2 or Fv fragments, andsingle-chain Fv (scFv) fragments that specifically bind to CatSper2protein or polypeptides, also can be used to inhibit CatSper2 activityby binding to extracellular domains of the protein and thereby block itsactivity.

Because most repressors or antagonists of CatSper2 expression oractivity will be reversible or will affect only mature sperm, theeffects of such compounds on fertility will be reversible because themolecules will be cleared from the body over time and new sperm areconstantly being produced. Thus, repressors or antagonists of CatSper2expression or activity can be used as human contraceptives because theycan cause reversible infertility. Such contraceptives can be takenorally or parenterally (e.g., injection, transdermal patch, orbioerodable implant) by females if they achieve sufficientconcentrations in the vagina, uterus or fallopian tubes to effectivelyinhibit CatSper2 activity and thereby decrease sperm motility or theability of sperm to penetrate the ZP. Similarly, such contraceptives canbe taken orally or parenterally by males if they achieve sufficientconcentration in the testes or seminal fluids to effectively inhibitCatSper2 expression or activity, and thereby decrease sperm motility orthe ability of sperm to penetrate the ZP. Alternatively, such compoundscan be formulated into lubricants, moisturizers, foams or jellies foruse with prophylactics, cervical caps, or contraceptive vaginal sponges,foams or jellies.

In another series of embodiments, repressors or antagonists of CatSper2genes and proteins can be used as contraceptives to treat non-humanmammals. These embodiments are similar to those described above. Suchcontraceptives can be used with respect to domesticated animals whichare maintained as pets, with respect to commercially valuabledomesticated animals (e.g., cows, sheep, horses), or with respect toanimal nuisances (e.g., mice, rats, raccoons, gophers). In someembodiments, the contraceptives are orally available and can be mixedinto food sources for the animals. In other embodiments, thecontraceptives can be administered parenterally (e.g., injection,transdermal patch, or bioerodable implant).

To the extent that the mammalian CatSper2 genes and proteins and thefish, amphibian and insect homologs of the CatSper2 genes and proteinsshare substantial sequence identity, repressors or antagonists ofmammalian CatSper2 genes and proteins can also be used in the control offish, amphibian and insect nuisances (e.g., mosquitoes). In addition,the non-mammalian homologs of the CatSper2 genes and proteins can beused to identify additional repressors and antagonists which are morespecific or effective for such homologs.

Methods of CatSper2 Genotyping and Diagnosing CatSper2-RelatedDisorders.

In another aspect, the present invention provides methods for genotypingsubjects with respect to the CatSper2 gene, and diagnosingCatSper2-related disorders such as infertility. Thus, for example, theCatSper2 nucleic acids (or a portion thereof) of a subject can be testedto ascertain whether that subject's CatSper2 genotype includes anymutations in the sequences relative to wild-type. Of particularsignificance would be mutations which introduce termination orframe-shift mutations that prevent the production of functional CatSper2proteins. However, point mutations that cause decreased CatSper2activity can also be identified. Similarly, the antibodies of thepresent invention can be used to test the sperm of a subject todetermine the presence or level of CatSper2 proteins. Of particular notewould be an absence or significant decrease in the level of CatSper2protein. Point mutations, however, can also cause infertility and can bedetected by antibodies which are specific for epitopes including oraffected by the mutant sequences. Determination of a subject's CatSper2genotype can be used for generic or reproductive counseling, or fordiagnosing infertility that results from a CatSper2 defect.

To determine a subject's CatSper2 genotype, or for diagnosing aCatSper2-related disorder, the nucleic acids of the invention can beused as primers in polymerase chain reaction (PCR) (e.g., anchor PCR orRACE PCR), or ligase chain reaction (LCR) amplifications of thesubject's DNA/mRNA. See, e.g., U.S. Pat. No. 4,683,195 and U.S. Pat. No.4,683,202; Landegran et al. (1988), Science 241:1077-1080; Nakazawa etal. (1994), Proc. Natl. Acad. Sci. USA 91:360-364; and Abravaya et al.(1995), Nucleic Acids Res. 23:675-682. Other useful methods foramplifying a subjects DNA/mRNA using the nucleic acids of the inventioninclude self-sustained sequence replication (e.g., Guatelli et al.(1990), Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptionalamplification (e.g., Kwoh et al. (1989), Proc. Natl. Acad. Sci. USA86:1173-1177), and Q-Beta Replicase-based systems (e.g., Lizardi et al.(1988), Bio/Technology 6:1197. The presence, absence or size of theresulting amplification products (e.g., Saiki et al. (1986), Nature324:163; Saiki et al. (1989), Proc. Natl. Acad. Sci. USA 86:6230; Gibbset al. (1989), Nucleic Acids Res. 17:2437-2448; Prossner (1993), Tibtech11:238; Gasparini et al. (1992), Mol. Cell Probes 6:1; Barany (1991)Proc. Natl. Acad. Sci. USA 88:189), direct sequencing of theamplification products (e.g., Maxim and Gilbert (1977), Proc. Natl.Acad. Sci. USA 74:560; Sanger (1977), Proc. Natl. Acad. Sci. USA74:5463), and other standard analytic techniques can be employed todetermine CatSper2 genotypes. The amplified products can also be used inmany of the techniques described below.

The nucleic acids of the invention also can be used as probes inhybridization and/or conformation-based assays to identify complementaryor imperfectly complementary sequences in a subject.

For example, in some embodiments, mutations can be identified byselectively hybridizing sample nucleic acids to immobilized controlnucleic acids. The controls can be adsorbed to filters or columns, orcan be arranged in high density ordered arrays containing hundreds orthousands of oligonucleotides probes (see, e.g., Cronin et al. (1996),Human Mutation 7:244-255; Kozal et al. (1996), Nature Medicine2:753-759).

In other embodiments, enzymatic or chemical cleavage can be employed tocleave or restrict duplexes of sample and control sequences atmismatched bases (e.g., Myers et al. (1985), Science 230:1242). Forexample, RNA/DNA duplexes can be treated with RNAse; DNA/DNA hybrids canbe treated with S1 nuclease to digest duplexes at mismatched bases; G/Amismatches are cleaved at the A by the E. coli mutY enzyme; and G/Tmismatches are cleaved at the T by the human thymidine DNA glycosylase(see, e.g., Hsu et al. (1994), Carcinogenesis 15:1657-1662). Chemicalcleavage of mismatches can be employed using, for example,hydroxylamine, osmium terroxide and/or piperidine. See generally, e.g.,Cotton et al. (1988), Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al.(1992), Methods Enzymol. 217:286-295; and U.S. Pat. No. 5,459,039.

In other embodiments, mutations can create or destroy specific sequenceswhich serve as cleavage points for restriction enzymes or ribozymes.Thus, restriction fragment length polymorphism (RFLP) analysis can beemployed in which (amplified) sample DNA is digested with at least onerestriction endonuclease, and the resulting fragment lengths areanalyzed and compared to controls to determine the presence or absenceof mutations which affect the pattern of restriction fragment lengths.Similarly, sequence-specific ribozymes can be used to identify mutationsthat create or destroy ribozyme cleavage sites. See, e.g., U.S. Pat. No.5,498,531.

In other embodiments, mutations can be detected by their effects on theelectrophoretic mobility of a sequence, either as a single-strandednucleic acid or as a duplex. For example, single-strand conformationpolymorphism (SSCP) analysis (Orita et al. (1989), Proc. Natl. Acad.Sci. USA 86:2766; Cotton (1993), Mutat. Res. 285:125-144; Hayashi(1992), Genet. Anal. Tech. Appl. 9:73-79; and Keen et al. (1991), TrendsGenet. 7:5), denaturing gradient gel electrophoresis (DGGE) (Myers etal. (1985), Nature 313:495), and temperature gradient gelelectrophoresis (Rosenbaum and Reissner (1987), Biophys. Chem.265:12753) can be employed.

These and other methods of detecting mutations in the CatSper2 genes andproteins will be apparent to one of ordinary skill in the art based uponthe nucleic acid and protein sequences disclosed herein.

In Vitro Fertilization.

In another aspect, the present invention provides a method of in vitrofertilization of ova by sperm characterized by decreased CatSper2expression or activity. Because of the role of CatSper2 in theflagellum, CatSper2-deficient sperm will be compromised in theirmotility and, therefore, their ability to fertilize ova. In particular,proper flagellar function is necessary for sperm to penetrate the ZonaPellucida (ZP) for fertilization (Ren et al. (2001), supra). Thus, thepresent invention provides a method of in vitro fertilization forCatSper2-deficient males in which the sperms of such males are treatedto overcome the CatSper2 deficiency or are contacted with ova from whichthe ZP have been removed. Because other genetic deficiencies can resultin sperm which are incapable of penetrating the ZP, this method can beextended to other males having genetic deficiencies which affect spermmotility or ZP-penetration, or for which in vitro fertilizationpreviously has been unsuccessful using ova with intact ZPs.

Methods of Treating CatSper2-Mediated Infertility.

In another aspect, the present invention provides methods of treatinginfertility in CatSper2-deficient males, in which an enhancer ofCatSper2 expression or an agonist of CatSper2 activity is administeredto the subject. In other embodiments, gene or protein therapy can beemployed to provide the Catsper2 gene or protein to sperm (or spermprogenitors) which are deficient in the CatSper2 gene or protein. Forgene therapy, a genetic construct encoding a CatSper2 protein can beemployed to cause expression of a CatSper2 protein in sperm or spermprogenitors which are deficient in the CatSper2 gene or protein.

In another aspect, infertility of a mating pair (e.g., a human couple)can result from antibodies generated by the female against antigenspresent on the sperm of the male. In some cases, the antibodies can bedirected against an epitope of a CatSper2 protein. Thus, the presentinvention also provides methods of diagnosing an anti-CatSper2antibody-mediated infertility caused by anti-CatSper2 antibodies presentin a female urogenital tract. The methods include obtaining a sample ofantibodies present in the female and contacting the antibodies withCatSper2 proteins or fragments of CatSper2 proteins. In someembodiments, the CatSper2 fragments are epitopes of the CatSper2proteins having high predicted antigenicity (e.g., approximatelyresidues 266-275, 386-400, 447-458, and 482-494 of SEQ ID NO: 2;approximately residues 66-99, 266-275, and 394-414 of SEQ ID NO: 4;approximately residues 64-89, 262-275 and 562-588 of SEQ ID NO: 6, andallelic and mammalian homologs thereof). In these methods, either thefemale's antibodies or the CatSper2 proteins/fragments optionally can beimmobilized and either the female's antibodies or the CatSper2proteins/fragments optionally can be detectably labeled to facilitatedetection of binding between the antibodies and the CatSper2proteins/fragments.

In these cases, administering an excess of the CatSper2 protein, or atleast a fragment of the CatSper2 protein including the relevant epitope,can saturate the binding sites of the anti-CatSper2 antibodies presentin the female's urogenital tract and thereby inhibit or reduce theantibody-mediated infertility. Alternatively, an anti-idiotypic antibody(i.e., an antibody which specifically binds to the variable regions ofanother antibody with a defined specificity) can be employed. That is,an antibody which binds specifically to anti-CatSper2 antibodies can beemployed to inhibit the anti-CatSper2 antibodies present in the female'surogenital tract and thereby inhibit or reduce the antibody-mediatedinfertility. One of ordinary skill in the art can easily identify therelevant CatSper2 epitopes recognized by such female antibodies (e.g.,using the methods described above) and produce substantially purepreparations of the relevant epitope or anti-idiotypic antibodies bystandard means. Thus, the invention also provides methods for treatingan anti-CatSper2 antibody-mediated infertility caused by anti-CatSper2antibodies present in a female urogenital tract. The methods includeadministering into the urogenital tract of the female an amount of therelevant CatSper2 epitope (or whole CatSper2 protein) or an amount ananti-idiotypic antibody effective to inhibit the anti-CatSper2antibodies and thereby inhibit or reduce the antibody-mediatedinfertility.

Business Methods Relating to CatSper2.

In another aspect, the present invention provides a method of conductinga drug discovery business comprising: identifying, by the assays of theinvention, one or more agents which antagonize CatSper2 activity;determining if an agent identified in such an assay, or an analog ofsuch an agent, inhibits at least one of sperm motility or eggpenetrance; conducting therapeutic profiling of an agent identified asan antagonist for efficacy and toxicity in one or more animal models;and formulating a pharmaceutical preparation including one or moreantagonist agents identified as having an acceptable therapeuticprofile.

In one embodiment, the drug discovery business further includes the stepof establishing a system for distributing the pharmaceutical preparationfor sale, and can optionally include establishing a sales group formarketing the pharmaceutical preparation.

In another aspect, the present invention provides a method of conductinga drug discovery business comprising: identifying, by the subject assay,one or more agents which agonize CatSper2 activity; determining if anagent identified in such an assay, or an analog of such an agent,increases at least one of sperm motility or egg penetrance; conductingtherapeutic profiling of an agent identified as an agonist for efficacyand toxicity in one or more animal models; and formulating apharmaceutical preparation including one or more agents identified ashaving an acceptable therapeutic profile.

In certain embodiments, the drug discovery business further includes thestep of establishing a system for distributing the pharmaceuticalpreparation for sale, and can optionally include establishing a salesgroup for marketing the pharmaceutical preparation.

In certain embodiments, the assay to identify agents which agonizeCatSper2 activity is conducted using wild type CatSper2. In anotherembodiment, the assay to identify agents which agonize CatSper2 activityis conducted using a mutant CatSper2. By a “mutant CatSper2” is meant aCatSper2 polypeptide containing one or more amino acid insertions,deletions, or substitutions, wherein said insertions, deletions, orsubstitutions change the amino acid sequence and activity of the mutantCatSper2 in comparison to wild type CatSper2. Such a change in activitycan include, but is not limited to, a change in motility, eggpenetrance, or cation transport. A change in activity could also includea change in the proper localization or expression of the CatSper2protein or mRNA.

In still another aspect, the invention provides a method of conducting areproductive medicine business comprising: examining a sperm sample froma male patient, wherein said patient is experiencing a fertilityproblem; determining if said sperm are characterized by at least one ofa decrease in motility or a decrease in egg penetrance; performing invitro analysis to determine the efficacy of a CatSper2 agonist inincreasing at least one of sperm motility or egg penetrance;establishing a treatment regimen comprising administering an amount of aCatSper2 agonist effective to increase at least one of sperm motility oregg penetrance by sperm from the male.

In certain embodiments, the method further includes a step wherein themale patient is monitored by a physician to evaluate improvement infertility. Such evaluation can include examination of sperm at regularintervals following the initiation of treatment to measure improvementsin one or more of sperm motility or egg penetrance. The frequency offollow-up evaluation by the treating physician will be determined by thephysician or a trained health care provider. Factors to consider are thepatient's schedule and comfort level, as well as the urgency with whicha male patient is attempting to father an offspring. Representativefollow-up appointments can be conducted weekly, semi-weekly, or monthly.In another embodiment, the method further includes the step of billingthe patient or the patient's insurance provider. In cases where thepatient's health insurance is paying for all or a portion of thefertility treatments, the policies of the health insurance provider willlikely influence the frequency of follow-up appointments.

In yet another aspect, the present invention provides a method ofconducting a contraceptive medicine business comprising: providing apharmaceutical preparation discovered through the methods of a drugdiscovery business, wherein said preparation inhibits the activity ofCatSper2; providing instructions to physicians and health care providersfor the administration of an amount of said pharmaceutical preparationeffective to inhibit the activity of CatSper2, wherein said effectiveamount is sufficient to prevent pregnancy.

In one embodiment, the method farther includes the step of establishinga system for distributing the pharmaceutical preparation for sale, andcan optionally include establishing a sales group for marketing thepharmaceutical preparation.

CatSper2 encodes a cation channel. Numerous types of cation channelsplay critical roles in cellular processes including regulation ofcardiac function (e.g., calcium channels). Thus, a great limitation ofmethods which employ administration of agents which either increase ordecrease the activity of cation channels is that such methods are likelyto have substantial side-effects. These side-effects can includesignificant cardiac complications, However, the results provided hereindemonstrate that CatSper2 is specifically expressed in sperm.Accordingly, agents which selectively increase or decrease the activityof CatSper2 can be administered to patients without the side effectsassociated with either general cation channel antagonists and agonists,or antagonists and agonists of cation channels which are more widelyexpressed in the body.

Through a drug discovery business, one or more agents which canantagonize the activity of CatSper2 can be identified. By antagonize theactivity is meant to decrease, in whole or in part, the activity ofCatSper2. Such a decrease in activity can be measured by examining atleast one of sperm motility, egg penetrance, or cation transport. Theterms decrease and antagonize will be used interchangeably throughout.

In certain embodiments, the initially identified CatSper2 agonist orantagonist can be subjected to further lead optimization, e.g., tofurther refine the structure of a lead compound so that potency andactivity are maintained but balanced with important pharmacologicalcharacteristics including: solubility, permeability, bioavailability,toxicity, mutagenicity, and pharmacokinetics (e.g., absorption,distribution, metabolism, elimination). Structural modifications aremade to a lead compound to address issues with these pharmacologicalparameters. These modifications however, most take into account possibleeffects on the molecule's potency and activity. For example, if thesolubility of a lead compound is poor, changes can be made to themolecule in as effort to improve solubility; these modifications,however, can negatively affect the molecule's potency and activity.Structure-activity relationship (SAR) data are then used to determinethe effect of the change upon potency and activity. Using an iterativeprocess of structural modifications and SAR data, a balance is createdbetween these pharmacological parameters and the potency and activity ofthe compound.

Candidate antagonists, or combinations thereof, must then be tested forefficacy and toxicity in animal models. Such therapeutic profiling iscommonly employed in the pharmaceutical arts. Before testing anexperimental drug in humans, extensive therapeutic profiling (e.g.,preclinical testing) must be completed to establish initial parametersfor safety and efficacy. Preclinical testing establishes a mechanism ofaction for the drug, its bioavailability, absorption, distribution,metabolism, and elimination through studies performed in vitro (that is,in test tubes, beakers, petri dishes, etc.) and in animals. Animalstudies are used to assess whether the drug will provide the desiredresults. Varying doses of the experimental drug are administered to testthe drug's efficacy, identify harmful side-effects that may occur, andevaluate toxicity.

Briefly, one of skill in the art will recognize that the identificationof a candidate agent which antagonizes CatSper2 activity in a drug-basedscreen is a first step in developing a pharmaceutical preparation usefulas a contraceptive agent. Administration of an amount of saidpharmaceutical preparation effective to successfully prevent pregnancy(e.g., to act as a useful contraceptive agent) must be both safe andeffective. Early stage drag trials, routinely used in the art, help toaddress concerns of the safety and efficacy of a potentialpharmaceutical. In the specific case of a CatSper2 antagonist, efficacyof the pharmaceutical preparation could be readily evaluated in a mouseor rat model. Briefly, male mice could be administered varying doses ofthe pharmaceutical preparations over various time schedules. Controlmale mice can be administered a placebo (e.g., carrier or excipientalone). The male mice are then allowed to mate freely by placing themales into cages with female mice, and measuring the rate of conceptionover time. Given the efficacy of currently available forms of birthcontrol, an effective contraceptive should be at least 80%, 85%, 90%,95%, 99%, or greater than 99% effective in preventing pregnancy.

In one embodiment, the step of therapeutic profiling includes toxicitytesting of compounds in cell cultures and in animals, analysis ofpharmacokinetics and metabolism of the candidate drug; and determinationof efficacy in animal models of diseases. In certain instances, themethod can include analyzing SARs and optimizing lead structures basedon efficacy, safety and pharmacokinetic profiles. The goal of such stepsis the selection of drug candidates for pre-clinical studies to lead tofiling of Investigational New Drug (“IND”) applications with the U.S.FDA and/or similar applications with similar regulatory authoritiesprior to human clinical trials.

Between lead optimization and therapeutic profiling, one goal of thesubject method is to develop a CatSper2 agonist or antagonist which hasminimal side-effects. In the case of antagonists, the lead compoundswill have clinically acceptable effects on vasodilatation (i.e.,dizziness, hypotension, headache, flushing, edema, etc.), myocardialischemia, hypotension, bradycardia, transient asystole, exacerbation ofheart failure, ventricular dysfunction, SA node or AV conductiondisturbances, or plasma digoxin levels.

By “toxicity profiling” is meant the evaluation of potentially harmfulside-effects which may occur when an effective amount of apharmaceutical preparation is administered. A side-effect may or may notbe harmful, and the determination of whether a side effect associatedwith a pharmaceutical preparation is an acceptable side effect is madeduring the regulatory approval process. Acceptable side effects vary dueto factors including: (a) the severity of the condition being treated,and (b) the availability of other treatments and the side-effectscurrently associated with these other treatments. For example, the termcancer encompasses a complex family of disease states related tomis-regulated cell growth, proliferation, and differentiation. Manyforms of cancer are particularly devastating diseases which cause severepain, loss of function of the effected tissue, and death.Chemotherapeutic drugs are an important part of the standard therapy formany forms of cancer. Although chemotherapeutics themselves can haveserious side-effects including hair-loss, severe nausea, weight-loss,and sterility, such side-effects are considered acceptable given theseverity of the disease they aim to treat.

In contrast, however, most currently available forms of birth control donot have significant side-effects. Thus, a pharmaceutical preparation ofa CatSper2 antagonist should have minimal toxicity and side-effects.Toxicity tests can be conducted in tandem with efficacy tests, and malemice administered effective doses of the pharmaceutical preparation canbe monitored for adverse reactions to the preparation. Potential adversereactions associated with a contraceptive agent may include loss of sexdrive, and behavioral changes. Blood, urine, and fecal samples takenfrom treated mice can also be monitored to detect any potential adversechanges in immune, kidney, or liver function. Additionally, given thatCatSper2 is a cation channel, mice receiving said pharmaceuticalpreparation should also be monitored for any changes in cardiac functionindicative of cross reactivity of the CatSper2 antagonist with othercation channels.

Agents which antagonize CatSper2 activity, and which are proven safe andeffective in animal studies, can be formulated into a pharmaceuticalpreparation. Such pharmaceutical preparations can then be marketed,distributed, and sold as contraceptive agents.

Given the link between loss of CatSper2 activity and fertility, there issubstantial utility in agents which increase the activity of CatSper2 totreat male fertility problems. Many instances of infertility involveproblems linked to the male. Such male infertility issues include lowsperm count, poor sperm motility, and abnormal sperm morphology.Currently there are few effective treatments for male-associatedinfertility.

The first step in developing potentially successful treatments for maleinfertility is the identification of CatSper2 agonists. A CatSper2agonist is one or more agents which increase the activity of CatSper2.As outlined in detail above for CatSper2 antagonists, agonists of theCatSper2 protein are expected to have fewer potential side-effects thanother cation channel agonists.

Methods for identifying agents which act as CatSper2 agonists areperformed largely as detailed for CatSper2 antagonists. However, auseful CatSper2 agonist will increase one or more of sperm motility oregg penetrance. Additionally, when identifying a CatSper2 agonist, suchan agent can agonize the activity of a wild type CatSper2. In addition,or alternatively, such an agent can agonize the activity of a mutantCatSper2. One or more agonists identified by these methods can then betested for safety and efficacy, as outline in detail above. Agents whichare shown to be safe and effective in animal studies are formulated intoa pharmaceutical preparation.

CatSper2 agonists are not likely to be effective for treating all malefertility problems. However, it is expected that some undeterminedpercentage of male fertility problems will be amenable to treatmentusing agonists of CatSper2 function. For example, a certain percentageof male infertility which results in poor sperm motility is likely dueto mutations in CatSper2. Given that CatSper2 is expressed specificallyin sperm, males possessing such a mutation would be expected to havelittle or no additional medical problems, and this explains in part whyinfertility is often found in otherwise healthy men. Additionally, aCatSper2 agonist can improve sperm motility overall, and thus helpcompensate for poor sperm motility due to other unrelated causes.

Conducting a Reproductive Medicine Business.

A pharmaceutical preparation including one or more agents which agonizethe activity of a wild type or mutant CatSper2 can be useful inestablishing a reproductive medicine business which provides treatmentfor candidate male patients experiencing fertility difficulties. Spermsamples provided by male patients are examined to determine ifinfertility in the male patients is amenable to treatment with thepharmaceutical preparation. Patients whose sperm is characterized by adecrease in at least one of motility or egg penetrance can be eligiblefor treatment. Prior to treatment, sperm samples provided by the malepatients are tested in vitro with the pharmaceutical preparation tofurther assess whether the male is eligible for treatment. Thisadditional step of in vitro testing helps to alleviate unnecessarytreatment in males whose infertility is unlikely to be improved with theCatSper2 agonist.

Male patients whose sperm shows increased motility or egg penetrance invitro are eligible for fertility treatment including the pharmaceuticalpreparation including one or more CatSper2 agonist. The exact treatmentregimen will vary from patient to patient, and can be readily determinedby an experienced medical professional. However, the treatment regimenwill include administration of an amount of said pharmaceuticalpreparation effective to increase at least one of sperm motility or eggpenetrance in said treated male. In certain embodiments, the increase insperm motility or egg penetrance will result in an increase infertility.

Pharmaceutical Preparations.

In another aspect, the invention provides pharmaceutically acceptablepreparations comprising a therapeutically effective amount of one ormore of the identified agents (i.e., antagonists or agonists) describedabove, formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention can be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscularor intravenous injection as, for example, a sterile solution orsuspension; (3) topical application, for example, as a cream, ointmentor spray applied to the skin; or (4) intravaginally or intrarectally,for example, as a pessary, cream or foam. However, in certainembodiments the subject compounds can be simply dissolved or suspendedin sterile water.

The phrase “therapeutically effective amount” as used herein means thatamount of an agent or composition which is effective for producing somedesired therapeutic effect, at a reasonable benefit/risk ratioapplicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject antagonistsfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes: (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations. In certain embodiments, the pharmaceutical preparation isnon-pyrogenic, i.e., does not elevate the body temperature of a patient.

The term “pharmaceutically acceptable salts” in this respect, refers tothe relatively non-toxic, inorganic and organic acid addition salts ofcompounds of the present invention. These salts can be prepared in situduring the final isolation and purification of the compounds of theinvention, or by separately reacting a purified compound of theinvention in its free base form with a suitable organic or inorganicacid, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. (See, for example, Berge et al.(1977), J. Pharm. Sci, 66:1-19.)

The pharmaceutically acceptable salts of the subject agents include theconventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfarnic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the agents of the present invention can contain one ormore acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of theagents, or by separately reacting the purified agent in its free acidform with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al. (1977), supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetra-acetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations can conveniently bepresented in unit dosage form and can be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, this amount will range from about 1% to about 99% of activeingredient, from about 5% to about 70%, or from about 10% to about 30%.

Methods of preparing these formulations or compositions include the stepof bringing into association one or more agents of the present inventionwith the carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association one or more agents of the present inventionwith liquid carriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product.

Formulations of the invention suitable for oral administration can be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. An agent of the presentinvention can also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the pharmaceuticalcompositions can also comprise buttering agents. Solid compositions of asimilar type can also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

A tablet can be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets can be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets can be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They can also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They can be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions can also optionally containopacifying agents and can be of such a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulared form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms can contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, can contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration can be presented as a suppository,which can be prepared by mixing one or more agents of the invention withone or more suitable nonirritating excipients or carriers comprising,for example, cocoa butter, polyethylene glycol, a suppository wax or asalicylate, and which is solid at room temperature, but liquid at bodytemperature and, therefore, will melt in the rectum or vaginal cavityand release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include peccaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of one ormore agents of this invention include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches and inhalants. Theactive agents can be mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels can contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of an agent of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the subject compound inthe proper medium. Absorption enhancers can also be used to increase theflux of the subject agent across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which can be reconstituted into sterileinjectable solutions or dispersions just prior to use, which can containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which can beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions can also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of macroorganisms can be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It can also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form can be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5%, or 0.5to 90%, of active ingredient in combination with a pharmaceuticallyacceptable carrier.

The following examples illustrate some specific modes of practicing thepresent invention, but are not intended to limit the scope of theclaimed invention. Alternative materials and methods can be utilized toobtain similar results.

EXAMPLE 1

Preparation of cDNA Library.

An enriched spermatid cell fraction from mouse 129Sv/Ev adult testes wasprepared using the unit gravity sedimentation method of Bellve (1993),Methods Enzymol. 225:84-113. Poly A⁺ RNA from the preparation wasreverse transcribed with random primers to synthesize double-strandedcDNA according to the suppliers protocol (Life Technologies/InvitrogenCorp., Carlsbad, Calif.). This cDNA (2 μg) was then subjected tosuppression subtraction hybridization (PCR Select™, Clontech, Palo Alto,Calif.) using driver cDNA (2 μg) prepared from a mixture of equalamounts of poly A⁺ RNA from nine different enriched tissues includingbrain, heart, intestine, kidney, liver, lung, skeletal muscle, spleen,and stomach. The resulting cDNA sample was subcloned into the signalpeptide trapping vector Notl site following digestion with EagI, and wastransformed into XL-10 Gold competent bacteria (Stratagene, La Jolla,Calif.) for amplification.

EXAMPLE 2 Signal Peptide Trapping.

A. Vector Preparation. The Saccharomyces cerevisiae invertase gene(Genbank Accession No. NC_001141.1, nucleotides 36484-37357 and37448-39483 including the promoter, coding sequence without the signalpeptide, and termination signals) was subcloned as an EcoRI/Sallfragment from pRB576 into the pBluescript™ plasmid (Stratagene, LaJolla, Calif.). The invertase coding sequence was modified bysite-directed mutagenesis (Quikchange, Stratagene, La Jolla, Calif.) toreplace the initiation methionine codon of the cytoplasmic enzyme formwith an alanine, and an artificial linker containing NotI and SalIcloning sites, was ligated into the HindIII - Smal site at the start ofthe invertase coding sequence. This modified invertase gene wassubcloned into the yeast shuttle, vector pYEUra3 (Genbank Accession No.U02457) using the EcoRI and Xhol restriction sites to produce pSPT IB.

B. Yeast Transformation and Selection. Signal peptide trapping wasperformed essentially as described by Klein et al. (1996), Proc. Natl.Acad. Sci. USA 93:7108-13. Briefly, the yeast strain YT455 (suc2×9,ade2-101, ura3-52) was transformed with the enriched spermatid cDNAlibrary using lithium acetate. The resulting transformants were selectedon minimal medium/-Ura dropout plates for three days at 30° C. and thenreplica plated onto YPS plates (2% sucrose as the sole carbon source) toselect for cDNA that encoded functional signal peptides. Those yeastcolonies that grew on sucrose over 7 days were analyzed for cDNAinserts.

C. Sequence analysis. Plasmids were released from yeast minicultures bylysis with SDS and glass beads (Strathern and Higgins (1991), MethodsEnzymol. 194:319-29). The cDNA inserts were amplified by PCR usingvector-specific primers flanking the cloning site. The resulting PCRproducts were subjected to automated sequencing followed by BLASTanalysts against the GenBank database (Altschul et al. (1997), NucleicAcid Res. 25:3389-3402).

D. Identification of CatSper2 Clone. An enriched spermatid cDNA librarywas screened using a yeast-based signal peptide selection method (Kleinet al. (1995), supra; Jacobs et al. (1997), Gene 198:289-96.). 1.2×10⁶colonies containing mouse spermatid cDNAs were screened. Approximately350 revertant yeast colonies were obtained after selection on sucrose.Of these selected cDNA clones, several known membrane/secreted proteinsspecifically expressed by sperm were identified (e.g., sp56, ferrilin β,ADAM 26, TPX-1) demonstrating the validity of the technique. A novelcDNA sequence encoding a protein with a deduced topology similar to thevoltage-gated potassium channels but containing a pore with calciumselectivity was identified and subsequently designated CatSper2.

EXAMPLE 3

Identification of Complete CatSper2 cDNA Sequences.

The complete coding sequence of the clone was determined using cDNAfragment-specific antisense and sense primers for 5′ and 3′ RACEamplifications, respectively (Marathon Ready Mouse™ Testis cDNA,Clontech, Palo Alto, Calif.). An intact full-length clone was producedusing pCR with a specific 5′ and 3′ UTR primer pair. The resulting PCRproduct was subcloned into pCR 2.1 (Stratagene, La Jolla, Calif.) andsequenced to verify the absence of mutations.

A full-length cDNA containing 1986 bp was isolated using RACE. Thepredicted ORF encodes a 588 amino acid protein with six transmembranesegments (FIG. 1(A)). The fourth transmembrane segment contains basicamino acid residues (K/R) spaced at every fourth position,characteristic of voltage-gated ion channels. A BLAST comparison of thissequence with the Genbank database revealed similarity to thevoltage-gated calcium channel family (Ca_(v)). The Na_(v), K_(v), cyclicnucleotide-gated (CNG), transient receptor potential (TRP), andpolycystin channels appeared more distantly related. Among the knownproteins, CatSper2 is most similar to CatSperl, another spermcell-specific putative cation channel recently found important formotility and normal fertilization (FIG. 1(B); Ren et al. (2001), supra).These two proteins are 21% identical and 40% similar within thetransmembrane region. The selectivity of ion channels is determined bythe pore-forming residues located between the fifth (S5) and sixth (S6)transmembrane segments. As shown in FIG. 1(B), several amino acidresidues in the pore (P) region are conserved between CatSper2,CatSperl, and the Cav channels including a critical aspartic or glutamicacid residue that defines calcium selectivity (Yaradi et al. (1999),Crit. Rev. Biochem. Mol. Biol. 34:181-214). The cytoplasmic portion ofCatSper2 following the transmembrane region contains additionalpotential functional motifs. The repetitive sequence region containsmultiple candidate tyrosine phosphorylation sites suggesting thatCatSper2 may represent one of the proteins phosphorylated duringcapacitation in the mouse (Visconti et al. (1995), Development121:1129-37). The C-terminal cytoplasmic region also contains anunconventional leucine zipper motif from residues 547-568 (Marx et al.(2001), J. Cell Biol. 153:699-708). This region may mediateprotein-protein interactions that form the actual channel pore complex,or that modulate CatSper2 channel activity in sperm cells.

Using the mouse CatSper2 sequence, three similar cDNA sequences encodingproteins with the same predicted topology as CatSper2 were cloned fromhuman testis. The proteins encoded by the human clones are 63-67%identical overall with mouse CatSper2. Two of the human clones encodepredicted ORFs of 528 (SEQ ID NO: 2) and 530 amino acids and differ fromeach other only by the presence of two tandem serine residues 54 ammoacid residues after the transmembrane region (i.e., two serine residuesare inserted between residues 392 and 393 of SEQ ID NO: 2). The thirdhuman clone encodes a predicted ORF of 414 amino acids (SEQ ID NO: 4),identical to the other human clones until amino acid residue 393, wherea 211 nucleotide gap in the cDNA sequence causes a frameshaft and earlytermination. The transmembrane region of all three human proteins is 77%identical to mouse CatSper2, including an identical P region. Thecytoplasmic region before the transmembrane segments and the carboxyterminal 63 amino acids of CatSper2 are also conserved in the two longerhuman homologs, 73% and 90% identical, respectively.

EXAMPLE 4 Northern Blot Analysis.

Total RNA (15 μg) from several mouse tissues and poly A⁺ RNA (2 μg) frommouse, rat, and human testes were separated on 1% agaroseMOPS/formaldehyde gels (Sambrook et al. (1989), supra). Followingtransfer to positively charged nylon membranes, the blots werehybridized overnight at 42° C. with either a random-primed ³²P probecorresponding to nucleotides 113 - 531 of the cDNA sequence (mousetissue blot) or nucleotides 919-1299 (cross-species blot: mouse, rat,and human) at 10⁶ cpm/ml in ULTRAhyb™ (Ambion, Inc., Austin, Tex.). Theblot was washed with 0.1×SSC/0.2% SDS/0.1% NaPP_(i) (1×15 min at roomtemperature, 3×15 min. at 65° C.) and exposed to film.

Northern blot analysts with a CatSper2 cDNA probe detected a single 2.1kb transcript in mouse testis. No signal was detected in other tissuesexamined (brain, heart, intestine, kidney, liver, ovary, skeletalmuscle, spleen, stomach), even with a sensitive RT-PCR assay. Thisresult, along with the presence of in-frame stop codons upstream of theputative initiation methionine, indicates that the transcript isfull-length. CatSper2 mRNA also was identified as a 2.0-2.1 kbtranscript in both rat and human testis samples.

By in situ hybridization of testicular tissue, it was shown that meioticand post-meiotic cells contained the CatSper2 transcript.Paraformaldehyde-fixed, paraffin-embedded mouse testis sections wereprobed with ³⁵S labeled CatSper2 sense or antisense probes (nucleotides113-531) and examined by bright field and dark field microscopy. Theantisense probe hybridized to the seminiferous tubules over bothspermatocytes and spermatids. There were no positive signals in othercells, and the sense probe also failed to hybridize to any testicularcell type.

EXAMPLE 5 In Situ Hybridization.

The region corresponding to nucleotides 113-531 was amplified by PCR andsubcloned into pCR 4.0-TOPO (Invitrogen Corporation, San Diego, Calif.).Radiolabeled (³⁵S) cRNA sense and antisense probes were transcribed fromthe linearized plasmid (MAXIscript™, Ambion, Inc., Austin, Tex.). Insitu hybridization of adult mouse testis sections was performedaccording to Shelton et al. (2000), J. Lipid Res. 41:532-7.

EXAMPLE 6 Production of Peptide Antibody.

Antibodies were made to synthetic peptides corresponding to amino acidresidues 64-89 and residues 562-588 of SEQ ID NO: 6 with a cysteineattached to the N-terminus. These peptides were conjugated tomaleimide-activated KLH according to the manufacturer's protocol (PierceChemical Co., Rockford, Ill.). Rabbits were immunized (intramuscularinjection) and subsequently boosted at regular intervals with 100 μg ofKLH-peptide conjugate per injection. Anti-peptide antibodies wereaffinity purified on the corresponding peptide chromatographic column(Sulfolink™, Pierce Chemical Co., Rockford, Ill.) using Gentle Bindingand Elution™ buffers (Pierce Chemical Co., Rockford, Ill.).

EXAMPLE 7 Immunoprecipitation and Immunoblotting.

For immunoprecipitation, samples were extracted with TBS (20 mMTris-HCl, 150 mM NaCl, pH 7.4) containing either 1% TX-100 or 1%Zwittergent 3-14 for at least 4 hours at 4° C. The extract wascentrifuged at 100,000x g for 30 min, and the resultant supernatantsolution was incubated for 2 hours with preimmune IgG covalently boundto Protein A agarose (Seize X™, Pierce Chemical Co., Rockford, Ill.).The nonbound fraction was recovered and incubated with peptide reactiveantibody (±1.5 μM peptide preincubation)/Protein A agarose overnightwith rocking at 4° C. The resulting immune complexes were washed 5times. SDS-polyacrylamide electrophoresis was according to Laemmli(1970), Nature 227:680-5. Electrophoretically separated samples weretransferred to nitrocellulose membranes according to Towbin et al.(1979), Proc. Natl. Acad. Sci. USA 76:4350-4, at 50V for 2 hours, 4° C.Immunoblots were probed in TBST/2.5% nonfat milk with CatSper2 antibody(1:5000) or CatSperl (1:2000) antibody. After incubation with theprimary antibody, the blots were rinsed once with TBST/2.5% nonfat milkand washed with TBST (4 changes over 30 min). Blots were then incubatedwith secondary goat anti-rabbit IgG-HRP conjugate for 1 hour, washedwith TBST (3 changes over 30 min), rinsed with TBS (3 times), and thesignal detected with chemiluminescence (Pierce Chemical Co., Rockford,Ill.).

Both CatSper2 and CatSperl are relatively insoluble when detergentsother than SDS are used to extract epididymal sperm. The proteins weremore easily solubilized from the testis with either 1% TX-100 orZwittergent 3-14. While CatSper2 was immunoprecipitated from both ofthese detergent extracts with the C-terminus peptide antibody, CatSperlremained in the soluble fraction. CatSper2 and CatSperl could not besolubilized with CHAPS or other detergents.

EXAMPLE 8 Immunolocalization (Fluorescence).

Caudal epididymal sperm cells were spotted onto glass slides and airdried. The cells were fixed and permeabilized with ice-cold methanol for2 min. The slides were rinsed in ethanol, air dried, and incubated withPBS/10% normal goat serum in CAS solution (Zymed Laboratories, Inc.,South San Francisco, Calif.) for 30 min to block nonspecific binding.The slides were then incubated in primary antibody diluted in blockingsolution (C-terminus antibody (1:5000); polyclonal antibody (1:1000)).Primary antibody was preincubated with either 20 μM competing peptide ora scrambled peptide prior to dilution in blocking solution to assessspecific binding. Following the primary antibody incubation, the slideswere washed with PBS (3×10 min), and incubated with goat anti-rabbitIgG-AlexaFluor-488 (Molecular Probes, Eugene, Oreg.) for 1 hr. Afterwashing with PBS (3×10 min), the slides were mounted with Fluoromount G(Fisher Scientific, Pittsburgh, Pa.) for observation.

Two peptide-reactive antibodies were produced as described above. SDSpolyacrylamide gel electrophoresis / immmmunoblots of mouse testis andcrude sperm membrane SDS extracts with each affinity-purified antibodyidentified a 68,000 Mr protein corresponding to the size predicted fromthe cDNA sequence. Similarly, a 68,000 Mr protein was detected in ratepididymal sperm cell membranes. Indirect immunofluorescence wasperformed on methanol-fixed, permeabilized mouse cauda epididymal spermlabeled with C-terminal peptide antibody. Bound antibody was detectedwith AlexaFluor-488 conjugated secondary antibody using phase contrastand epifluorescence microscopy. The CatSper2 protein localized to theflagellum. This signal was blocked by preincubation with a competingpeptide, but not with a scrambled version of the peptide.

EXAMPLE 9 Fluorescence In Situ Hybridization.

A mouse genomic clone was identified with the cDNA probe correspondingto nucleotides 113-531 of SEQ ID NO: 5. The BAC clone was labeled withdigoxigenin dUTP by nick translation and used to probe normal metaphasechromosomes from mouse embryo fibroblast cells in 50% formamide, 10%dextran sulfate, and 2×SSC. Specific hybridization signals were detectedwith fluoresceinated anti-digoxigenin antibodies and the chromosomescounterstained with DAPI. A total of 80 metaphase cells were analyzedwith specific labeling detected in each case (Incyte Genomics, Inc.,Palo Alto, Calif.).

The CatSper2 gene is located on mouse chromosome 2 E5-F1. No reportedmutations associated with male infertility are present in this region ofthe mouse genome. In the human, two genes with 70-75% identity toCatSper2 at the nucleotide level were identified on chromosome 15q13, aregion that is syntenic with the mouse chromosome location of CatSper2.All three of the human testis clones described above appear to betranscribed from only one of these genes.

EXAMPLE 10 Electrophysiology.

CatSper2, either alone, or together with CatSperl and/or cyclicnucleotide gated channel subunit (CNG4) were transfected into CHO-K1 andHEK-293 cells. Whole cell patch clamp recordings were done as previouslydescribed (Ren et al., (2001), supra). The pipette solution contained120 mM Cs⁺, 60 mM glutamic acid, 20 mM TEA-Cl, 5 mM MgCl2, 3 mM Mg-ATP,10 mM EGTA, 10 mM HEPES and 5 mM D-glucose at pH 7.4. The bath contained135 mM NaCl, 5 mM KCl, 10 mM CaCl2, 10 mM Na-lactate, 10 mM Na-pyruvate,10 mM glucose and 30 mM HEPES at pH 7.4.

In each case, an associated current elicited by changes in voltage, pH,osmolarity, and/or cyclic nucleotide concentration was not detected,indicating that CatSper2 alone does not form a functional ion channel inthese cells. Similarly, heterologous expression of CatSperl alone or incombination with CNG channel β subunits failed to produce a functionalion channel (Ren et al. (2001), supra). As CatSper1 and CatSper2 aremembers of a unique ion channel family with overlapping expressionpatterns, their ability to associate to form a functionalheterotetrameric channel was tested. Co-expression of these two proteinsalso failed to yield a functional channel.

Equivalents

While this invention has been particularly shown and described withreferences to certain embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details can bemade therein without departing from the spirit and scope of theinvention. Those skilled in the art will recognize, or be able toascertain using no mote than routine experimentation, many equivalentsto the specific embodiments of the invention described specificallyherein. Such equivalents are intended to be encompassed in the scope ofthe invention.

What is claimed is:
 1. A transgenic mouse comprising a modification intothe mouse, the modification comprising an insertion of a nucleic acidencoding a CatSper2 protein, the CatSper2 protein comprising at least90% similarity to the amino acid sequence of SEQ ID NO: 5, and whereinthe CatSper2 protein has CatSper2 activity.
 2. A transgenic mousecomprising a modification into the mouse, wherein the modification isreplacement of an endogenous CatSper2 protein sequence with a reporteror marker gene, wherein the CatSper2 protein comprises at least 90%similarity to the amino acid sequence of SEQ ID NO:
 5. 3. A transgenicmouse comprising a modification into the mouse, the modificationcomprising inactivation of an endogenous CatSper2 protein, wherein theCatSper2 protein comprises at least 90% similarity to the amino acidsequence of SEQ ID NO:
 5. 4. The transgenic mouse of claim 1, whereinthe modification is by homologous recombination to result in expressionof a reporter gene.
 5. The transgenic mouse of claim 1, wherein themodification is by homologous recombination to result in expression of amarker gene.
 6. The transgenic mouse of any one of claims 1-3, whereinthe CatSper2 sequences are conspecific to the transgenic mouse.
 7. Thetransgenic mouse of any one of claims 1-3, wherein the CatSper2sequences are transpecific to the transgenic mouse.